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Characterisation of protein dual targeting to energy
organelles in Arabidopsis thaliana
Chris Carrie
This thesis was submitted as part of the requirement for the degree of Doctor of
Philosophy at the University of Western Australia
December 2010
ARC Centre of Excellence in Plant Energy Biology
School of Biomedical, Biomolecular and Chemical Sciences
Chris carrie
II
“The price of success is hard work, dedication to the job at hand,
and the determination that whether we win or lose, we have
applied the best of ourselves to the task at hand.”
Vince Lombardi
Chris carrie
III
Declaration The research presented in this thesis is my own work unless otherwise stated. This work
was carried out in the Australian Research Council Centre of Excellence in Plant
Energy Biology at the University of Western Australia. The material presented in this
thesis has not been submitted for any other degree
Chris Carrie
Chris carrie
IV
Acknowledgements First and foremost I would like to thank my supervisor Jim for all his expertise,
guidance, and support over the past few years, and also for allowing me to follow some
abstract ideas, like seeing if ND proteins can target peroxisomes. I would also like to
thank all of the members, past and present, from the Whelan lab for making a great
working environment and for putting up with me even when I was a little grumpy.
Special mentions must go to Monika, Reena, and Estelle for not only providing your
expertise and knowledge for various parts of this thesis, but also for your friendship
throughout the course of my PhD. I want to thank everyone else from the Centre of
Plant Energy Biology, as it really has been a great place to come to work everyday.
Special mentions must go to Harvey, Etienne, and Holger for kindly giving me some of
your hard earned peroxisomes for use in some of my experiments.
I would like to thank my family who have supported me throughout this journey, even
when I missed family events to be in the lab. Part of this thesis really belongs to all of
you as well because without you I probably never would have been able to do it. I wont
mention everyone by name there really are to many of you but special must go to my
Mum. Lastly I would like to thank Amy for the unconditional love and support I get
from you everyday.
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Publications Primary studies published during my PhD: Study I: Carrie C, Murcha MW, Millar AH, Smith SM, Whelan J (2007) Nine 3-
ketoacyl-CoA thiolases (KATs) and acetoacetyl-CoA thiolases (ACATs) encoded by five genes in Arabidopsis thaliana are targeted either to peroxisomes or cytosol but not to mitochondria. Plant Mol Biol 63: 97-108.
Study II: Carrie C, Kuhn K, Murcha MW, Duncan O, Small ID, O'Toole N,
Whelan J (2009) Approaches to defining dual-targeted proteins in Arabidopsis. Plant J 57: 1128-1139.
Study III: Carrie C, Murcha MW, Kuehn K, Duncan O, Barthet M, Smith PM,
Eubel H, Meyer E, Day DA, Millar AH, Whelan J (2008) Type II NAD(P)H dehydrogenases are targeted to mitochondria and chloroplasts or peroxisomes in Arabidopsis thaliana. FEBS Lett 582: 3073-3079.
Study IV: Lister R, Carrie C, Duncan O, Ho LH, Howell KA, Murcha MW,
Whelan J (2007) Functional definition of outer membrane proteins involved in preprotein import into mitochondria. Plant Cell 19: 3739-3759.
Study V: Carrie C, Giraud E, Duncan O, Xu L, Wang Y, Huang S, Clifton R,
Murcha M, Filipovska A, Rackham O, Vrielink A, Whelan J (2010) Conserved and Novel Functions for Arabidopsis thaliana MIA40 in Assembly of Proteins in Mitochondria and Peroxisomes. J Biol Chem 285: 36138-36148
Study VI: Carrie C, Murcha MW, Whelan J (2010) An in Silico Analysis of the
Mitochondrial Protein Import Apparatus of Plants. BMC Plant Biol 10: 249
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Additional publications Albrecht V, Simkova K, Carrie C, Delannoy E, Giraud E, Whelan J, Small ID, Apel K,
Badger MR, Pogson BJ (2010) The Cytoskeleton and the Peroxisomal-Targeted SNOWY COTYLEDON3 Protein Are Required for Chloroplast Development in Arabidopsis. Plant Cell, in press
Carrie C, Giraud E, Whelan J (2009) Protein transport in organelles: Dual targeting of
proteins to mitochondria and chloroplasts. FEBS J 276: 1187-1195 Giraud E, Ng S, Carrie C, Duncan O, Low O, Lee CP, Van Aken O, Millar AH,
Murcha MW, Whelan J (2010) TCP transcription factors link the regulation of genes encoding mitochondrial proteins with the circadian clock in Arabidopsis thaliana. Plant Cell, in press
Ho LH, Giraud E, Lister R, Thirkettle-Watts D, Low J, Clifton R, Howell KA, Carrie C, Donald T, Whelan J (2007) Characterization of the regulatory and expression context of an alternative oxidase gene provides insights into cyanide-insensitive respiration during growth and development. Plant Physiol 143: 1519-1533
Jia L, Wu Z, Hao X, Carrie C, Zheng L, Whelan J, Wu Y, Wang S, Wu P, Mao C
(2010) Identification of a novel mitochondrial protein, short postembryonic roots 1 (SPR1), involved in root development and iron homeostasis in Oryza sativa. New Phytol, in press
Millar AH, Carrie C, Pogson B, Whelan J (2009) Exploring the function-location
nexus: using multiple lines of evidence in defining the subcellular location of plant proteins. Plant Cell 21: 1625-1631
Murcha MW, Elhafez D, Lister R, Tonti-Filippini J, Baumgartner M, Philippar K,
Carrie C, Mokranjac D, Soll J, Whelan J (2007) Characterization of the preprotein and amino acid transporter gene family in Arabidopsis. Plant Physiol 143: 199-212
Thatcher LF, Carrie C, Andersson CR, Sivasithamparam K, Whelan J, Singh KB
(2007) Differential gene expression and subcellular targeting of Arabidopsis glutathione S-transferase F8 is achieved through alternative transcription start sites. J Biol Chem 282: 28915-28928
Van Aken O, Zhang B, Carrie C, Uggalla V, Paynter E, Giraud E, Whelan J (2009)
Defining the Mitochondrial Stress Response in Arabidopsis thaliana. Mol Plant 2:1310-1324
Chris carrie
VII
Abbreviations ACAT Acetoacetyl-CoA thiolase
AGAT Alanine/glyoxylate aminotransferase
AGL Agamous like protein
AOX Alternative oxidase
APL Altered phloem development
CaS Calcium-sensing receptor
Ccs1 Copper/zinc chaperone for superoxide dismutase
CP Carrier protein
CSD Copper/zinc superoxide dismutase
Erv1 Essential for respiration and vegetative growth
ER Endoplasmic reticulum
FAD Flavin adenine dinucleotide
GDP Guanosine diphosphate
GeBP GL1 enhancer binding protein
GFP Green flouresence protein
GST Glutathione S-transferase
Hot13 Helper of Tim protein of 13 kDa
Icp55 Intermediate cleavage peptidase of 55 kDa
KAT 3-Ketoacyl-CoA thiolase
MCD Malonyl CoA decarboxylase
Mdm10 Mitochondria distribution and morphology protein 10
MIA Mitochondrial import and assembly
Mim1 Mitochondrial import 1
MPP Mitochondrial processing peptidase
ND Type II alternative NAD(P)H dehydrogenase
NDC1 Type II alternative NAD(P)H dehydrogenase C1
OM64 mitochondrial outer membrane protein of 64 kDa
Omp85 Outer membrane protein of 85 kDa
PRAT Preprotein and amino acid transporter
PQ Plastoquinone
PTS1 Peroxisomal targeting signal type 1
RFP Red flouresence protein
SPP Stromal processing peptidase
SSU Small subunit of Ribulose 1,5-bisphosphate carboxylase/oxygenase
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VIII
TLP Tubby like protein
TIC Translocase of the inner envelope of chloroplasts
TIM Translocase of the inner mitochondrial membrane
TOC Translocase of the outer envelope of chloroplasts
TOM Translocase of the outer mitochondrial membrane
TPR Tetratricopeptide repeat
SAM Sorting and assembly machinery
UTR Untranslated region
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Abstract Eukaryotic cells are defined by their containment of membrane bound
compartments, termed organelles. The majority of proteins found within a particular
organelle are encoded by genes in the nucleus, synthesised in the cytosol, and
subsequently targeted to specific organelles. The traditional view of biology is that one
gene gives rise to one protein, targeted to one location. However, in the past 15 years an
increasing number of proteins have been found to be localised in more than one
organelle, a phenomenon called dual targeting.
In the model plant Arabidopsis thaliana, only a limited number of proteins have
been identified to date as being dual targeted. The work carried out in studies I, II and
III aimed to identify new dual targeted proteins in Arabidopsis. A list of candidate dual
targeted proteins was defined by cross-referencing a number of publically available
subcellular localisation datasets, generated by large scale proteomic studies. In addition,
candidate genes were also identified by computational predictions, based on the protein
amino acid sequences. A selection of proteins were then selected for targeting analysis
by in vivo green fluorescent protein (GFP) tagging, results were then confirmed by
either in vitro import assays or Western blot analysis.
In this way, studies I, II, and III led to the identification of 12 new dual targeted
proteins in Arabidopsis. Five proteins were found to target both mitochondria and
plastids, one was found to target mitochondria and the nucleus, and five were found to
target both mitochondria and peroxisomes. The latter is particularly significant as this
was the first time that dual targeting between mitochondria and peroxisomes had been
demonstrated in plants. Of these, three of the alternative NAD(P)H dehydrogenases of
the inner mitochondrial membrane were subsequently found to also be targeted to the
peroxisome. This is mediated by an N-terminal mitochondrial targeting signal and a C-
terminal peroxisomal targeting signal. The peroxisomal targeting was missed in
previous studies due to the detection of localisation using only C-terminal GFP fusions.
By analysing the subcellular localisation of Arabidsis thiolase proteins, study I revealed
that the β-oxidation of fatty acids does not occur in plant mitochondria, given that no
thiolases were found to be targeted to mitochondria in Arabidopsis. This was in contrast
to previous proteomic and activity assays which determined that some plant thiolases
were mitochondrial. These results confirmed the requirement for multiple lines of
complimentary evidence when performing subcellular localisation studies.
Chris carrie
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While analysing the potential dual targeted proteins, it become evidenced that a
number of experimental parameters are critical for designing GFP based studies,
especially with regards to dual targeted proteins. It was shown that the location of the
passenger protein, whether it be C- or N-terminally based, the type of tissue used and
the analyses of all possible gene models are critical for accurate determination of
localisation (Study II). Furthermore, all results must also be verified by a second
technique, so that multiple lines of complementary data are used before subcellular
localisation is accurately determined. These guidelines were proposed after it was
discovered that a number of proteins previously assigned to only one location, were
subsequently found to be dual targeted after using these techniques. Also, some proteins
previously thought to be dual targeted were found to be only targeted to one organelle.
In order to better understand the mechanisms of dual targeting in Arabidopsis,
study IV aimed to determine the mitochondrial receptors involved in the import of dual
targeted proteins. It was demonstrated that dual targeted proteins appear to use a
different import pathway than mitochondrial specific proteins. This was proposed to be
mediated by the mitochondrial import receptor Metaxin, which was demonstrated to
interact with dual targeted proteins (Study IV). In addition, a new plant specific
mitochondrial import receptor was identified, OM64, (Study IV). Taking this further,
study VI analysed the mitochondrial import apparatus in a number of different plant
species and other organisms, revealing that the evolution of the mitochondrial import
apparatus of plants is still an on going process. The identified differences between plant
import components and their yeast and human counterparts is thought to be due to the
selective pressure to sort proteins between mitochondria and chloroplasts, suggesting a
novel mode of evolution in plants.
Whilst analysing the mitochondrial import components of Arabidopsis, it
became apparent that the protein orthologues to the yeast Mia40 in Arabidopsis
contained a peroxisomal targeting signal. Upon closer, in vivo analysis of Mia40
localisation, it was evidenced that in Arabidopsis, Mia40 is dual targeted to
mitochondria and peroxisomes, unlike the yeast Mia40, which is localised only in the
mitochondria (Study V). Upon functional analysis of Arabidopsis Mia40, it was also
found to be different to yeast, in that it was not required for the inner membrane space
disulfide relay cycle. It was shown that Arabidopsis Mia40 is involved in the formation
of complex I of the mitochondrial respiratory chain, and also in the oxidative folding of
Chris carrie
XI
the copper/zinc chaperone for SOD (Ccs1) and copper/zinc superoxide dismutase
(SOD), in both the mitochondria and peroxisomes. Thus, plants have gained an extra
function for Mia40, in addition to having it dual targeted to the peroxisomes and
mitochondria. These findings support the theory that proteins are dual targeted in order
to increase the functions of some genes.
Overall, a number of novel dual targeted proteins were identified in Arabidopsis,
not only between mitochondria and chloroplasts, but also between mitochondria and
peroxisomes, and mitochondria and the nucleus, giving novel insights into functions of
these proteins. A number of factors that can influence dual targeting were also shown,
including the type of tissues and techniques used. While the extent of dual targeting and
the exact mechanisms by which it occurs are only just beginning to be understood, the
reasons why some proteins are dual targeted are still largely unknown. However, by
utilising some of the techniques used in this thesis it may eventually possible to identify
all dual targeted proteins in plants, which upon closer inspection could give insights into
the purpose for dual targeting in plants
Chris carrie
XII
Contents
DECLARATION .......................................................................................................... III
ACKNOWLEDGEMENTS ......................................................................................... IV
PUBLICATIONS ........................................................................................................... V
ADDITIONAL PUBLICATIONS .............................................................................. VI
ABBREVIATIONS ..................................................................................................... VII
ABSTRACT .................................................................................................................. IX
CONTENTS ................................................................................................................. XII
CHAPTER 1 .................................................................................................................... 1
GENERAL INTRODUCTION ...................................................................................... 1
1.0 EUKARYOTIC CELL EVOLUTION – ENDOSYMBIOSIS .................................................. 2
1.1 GENE TRANSFER ....................................................................................................... 2
1.2 PEROXISOME – ENDOSYMBIOSIS VS NON-ENDOSYMBIOTIC ORIGIN ........................... 3
1.3 PROTEIN TARGETING AND IMPORT ............................................................................ 4
1.3.1 Plastid protein targeting and import ................................................................ 4
1.3.2 Plastid targeting signals .................................................................................. 4
1.3.3 Outer envelope membrane import machinery: TOC complex ......................... 5
1.3.4 Inner envelope import machinery: TIC complex ............................................. 7
1.3.5 Thylakoid protein import .................................................................................. 8
1.4 MITOCHONDRIAL PROTEIN TARGETING AND IMPORT ................................................ 8
1.4.1 Mitochondrial targeting signals ....................................................................... 8
1.4.2 Mitochondrial protein import .......................................................................... 9
1.5 PROTEIN IMPORT INTO PLANT MITOCHONDRIA ....................................................... 12
1.6 PEROXISOMAL PROTEIN IMPORT ............................................................................. 14
1.6.1 Receptor cargo interaction and membrane docking ...................................... 14
1.6.2 Receptor cargo translocation cargo release and receptor recycling ............ 15
1.7 DUAL TARGETING ................................................................................................... 17
1.7.1 Ambiguous targeting signals .......................................................................... 18
1.7.2 Alternative mechanisms of dual targeting ...................................................... 19
1.7.3 Systematic studies of dual targeted proteins .................................................. 20
1.8 RESEARCH PROPOSAL ............................................................................................. 21
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XIII
CHAPTER 2 .................................................................................................................. 26
FOREWORD TO STUDY I ......................................................................................... 27
CHAPTER 3 .................................................................................................................. 40
FOREWORD TO STUDY II ....................................................................................... 41
CHAPTER 4 .................................................................................................................. 54
FOREWORD TO STUDY III ...................................................................................... 55
CHAPTER 5 .................................................................................................................. 62
FOREWORD TO STUDY IV ...................................................................................... 63
CHAPTER 6 .................................................................................................................. 86
FOREWORD TO STUDY V ........................................................................................ 87
CHAPTER 7 .................................................................................................................. 99
FOREWORD TO STUDY VI .................................................................................... 100
CHAPTER 8 ................................................................................................................ 116
GENERAL DISCUSSION .......................................................................................... 117
7.1 DEFINING DUAL TARGETED PROTEINS: TARGETING VS ACCUMULATION STUDIES . 117
7.2 MECHANISMS OF DUAL TARGETING – SIGNALS AND IMPORT RECEPTORS .............. 122
7.3 REASONS FOR DUAL TARGETING ........................................................................... 124
7.4 FUTURE PERSPECTIVES ......................................................................................... 126
REFERENCES ............................................................................................................ 127
Chapter 1 General Introduction
1
Chapter 1
General Introduction
Chapter 1 General Introduction
2
1.0 Eukaryotic cell evolution – Endosymbiosis
The defining feature of eukaryotic cells is that they contain membrane bound
compartments termed organelles. Plant cells contain three organelles involved in energy
metabolism; plastids, mitochondria, and peroxisomes. Chloroplasts, (specialised
plastids) produce energy through the conversion of light energy into chemical energy.
Chloroplasts also synthesise amino acids, lipids, and many other specialised
compounds. Mitochondria are involved in energy metabolism through the oxidation of
organic acids into reduced nucleotides via the tricarboxcyclic acid cycle (TCA cycle),
which are finally oxidised into chemical energy by the electron transport chain.
Peroxisomes play an important role in plant energy metabolism in a number of ways,
such as lipid metabolism, photorespiration, nitrogen metabolism, detoxification, and the
synthesis of some plant hormones. Integral to the function of plant cells is the
integrated nature of metabolism, as all three organelles work together to produce the
energy required for cellular growth and maintenance (Siedow and Day, 2000).
Except for rare exceptions in prokaryotes, the metabolic compartmentalisation
of organelles is specific to eukaryotic cells (Martin, 2010). The endosymbiotic origin of
organelles is explained by the hypothesis that mitochondria and plastids were once free-
living bacteria that underwent evolutionary transformation into complex metabolic
compartments (Tielens et al., 2002; van der Giezen, 2009). Both mitochondria and
plastids have retained their own DNA and as such, the sequence and structure provides
compelling evidence that they were once free living prokaryotes (Gray, 1999).
However, these organellor genomes are highly reduced in comparison to their free-
living ancestors (Gray, 1999). Plastid genomes have been shown to encode between 20
– 200 proteins, ywhilst mitochondrial genomes encode for as little as 3 proteins in
Plasmodium falciparum, ranging up to 67 proteins in Reclinomonas americana (Timmis
et al., 2004). In the particular case of highly specialised mitochondria called mitosomes
and hydrogenosomes, organelles which have lost their entire genomes (Tovar et al.,
2003; Boxma et al., 2005; van der Giezen and Tovar, 2005; van der Giezen, 2009),
energy metabolism still occurs though they have lost their ability to carry out oxidative
phosphorylation.
1.1 Gene transfer
Despite the reduction in genome size, plastids and mitochondria still contain
more than one thousand proteins (Millar et al., 2006). During millions of years of
Chapter 1 General Introduction
3
evolution these organelles lost or transferred most of their genes to the nucleus (Martin
et al., 1993). The transferred genes acquired expression and targeting signals such that
the encoded protein could be translated on cytosolic ribosomes and imported into the
organelle to achieve function (Martin, 2010). Once the host copy of a particular gene
acquired all the mechanisms for correct expression, translation, import, and function,
the organelle copy was no longer required and was lost, thus completing endosymbiotic
gene transfer (Allen, 2003). It could readily be imagined that at the onset of
endosymbiosis, which led to the formation of mitochondria and plastids, the
endosymbiont may have underwent lysis, providing a pool of total ‘genome’ DNA, that
may have been subsequently incorporated into the host genomic DNA. This is referred
to as genome transfer. This type of genome transfer, or partial genome transfer, can be
observed even today between organelles and the nucleus (Timmis et al., 2004; Kleine et
al., 2009). However, it has yet to be shown that this results in functional gene relocation
between organelles and the nucleus. In contrast, both for mitochondria and plastids, the
transfer of individual genes likely occurs via a reverse transcribed cDNA from organelle
mRNA, a process shown to be still ongoing (Adams and Palmer, 2003).
1.2 Peroxisome – Endosymbiosis vs non-endosymbiotic origin
There are currently two theories to explain the origin of peroxisomes; the first is
that peroxisomes originate from an ancient endosymbiont, and the second suggests that
peroxisomes are derived from the endoplasmic reticulum (ER) (Gabaldon, 2010). The
fact that core peroxisomal mechanisms for division, biogenesis, and maintenance are
conserved across a diverse range of organisms (Gabaldon et al., 2006) suggests that
peroxisomes have likely evolved from a common ancestor arising from a single
evolutionary event. The endosymbiotic origin for peroxisomes was proposed after it
was realised that peroxisomes are formed by division and have the ability to import
proteins in a post-translational manner. These features are common with the
endosymbiotically derived mitochondria and plastids (Lazarow and Fujiki, 1985). An
endosymbiotic origin for peroxisomes provides an appealing metabolic scenario, which
takes into account the role of enzymes involved in the detoxification of highly reactive
oxygen species (de Duve, 1982). According to this theory, the proto-peroxisome was
acquired at a time when the level of atmospheric oxygen was increasing and represented
a toxic compound for the majority of organisms at the time (de Duve, 1982), proposing
that peroxisomes originated from an ancient actinobacterium (Duhita et al.).
Chapter 1 General Introduction
4
However there is considerable experimental evidence demonstrating that the
biogenesis of peroxisomes is tightly linked to the ER. Evidence supporting this proposal
include the demonstration that some peroxisomal membrane proteins are first targeted
to the ER prior to reaching peroxisomes. Additionally, peroxisome-less yeast mutants
can form peroxisomes de novo from the ER upon introduction of the wildtype gene
(Erdmann and Kunau, 1992; Tabak et al., 2003). Independent phylogenetic evidence
also links peroxisome evolution to the ER, showing that components of the peroxisomal
import machinery are related to the components of the ER associated decay pathway
(Gabaldon et al., 2006; Schluter et al., 2006). It should also be noted that some plastid
proteins are initially targeted via the ER, suggesting that the endosymbiotic evidence for
the origin of peroxisomes may have been lost over time (Villarejo et al., 2005).
1.3 Protein targeting and import
Irrespective of the evolutionary origin, the majority of organellor proteins are
encoded by the nucleus. These cytosolically synthesized proteins must be targeted to the
correct organelle (Figure 1.1). Organellor proteins contain within their amino acid
sequences all the information required for targeting to the correct organelle. A brief
description of plastid, mitochondrial, and peroxisomal targeting signals and import
apparatus is outlined below.
1.3.1 Plastid protein targeting and import
Most prior research on plastid protein import has been carried out using
chloroplasts, which as the site of photosynthesis, are the most abundant type of plastids.
It is proposed that other types of plastids contain the same import apparatus on the
envelope membranes (Strzalka et al., 1987; Wan et al., 1996; Davila-Aponte et al.,
2003). A general import pathway of the chloroplast envelopes has been described which
constitutes the translocons at the outer and inner envelope of chloroplasts (TOC and
TIC respectively) (Figure 1)(Balsera et al., 2009).
1.3.2 Plastid targeting signals
The majority of nuclear encoded chloroplast proteins contain an N-terminal
targeting signal, which is cleaved upon import by the stromal processing peptidase
(SPP) (Bruce, 2000, 2001). Early research into chloroplast targeting signals proposed
that they would contain definite motifs and structural features specifically designed to
avoid mis-targeting to other organelles. However, plastid targeting signals are in fact
Chapter 1 General Introduction
5
heterogeneous in sequence and are mostly unstructured (Bruce, 2000, 2001). The
general features of chloroplast targeting signals are that they lack acidic residues giving
an overall positive charge, they are enriched in hydroxylated amino acids (mainly
serine) and vary in length between 20 – 150 amino acids. These features also resemble
the targeting signals of mitochondrial proteins. In contrast to mitochondrial targeting
signals, chloroplast targeting signals show no secondary structure in aqueous solution
(Krimm et al., 1999). It has been proposed that chloroplast targeting signals can form a
perfect random coil (von Heijne and Nishikawa, 1991). The lack of a secondary
structure is thought to be due to the recruitment of cytosolic factors following
translation (May and Soll, 2000; Zhang and Glaser, 2002). It has also been observed
that some targeting signals acquire a typical structure upon contact with the lipid
environment of the outer envelope membrane, which has a unique composition that
distinguishes it from the outer membrane of mitochondria (Wienk et al., 2000; Bruce,
2001). The interaction between the targeting signals and the galactolipids specific to the
outer envelope membrane has been speculated to stimulate import (Chen and Li, 1998).
A recent study using hierarchical clustering of targeting signals defined motifs
contained within the targeting signals into seven distinct groups (Lee et al., 2008).
1.3.3 Outer envelope membrane import machinery: TOC complex
The TOC complex is composed of a channel protein (Toc75), two GTPase
receptors (Toc159 and Toc34), and two dynamically associated components (Toc64 and
Toc12) (Oreb et al., 2008). The import channel forming protein, Toc75, is a highly
conserved β-barrel protein which is evolutionary related to Omp85, a protein involved
in the integration of proteins into the bacterial outer membrane in gram negative
bacteria (Bolter et al., 1998; Gentle et al., 2005). The Toc75 protein contains two
domains; an N-terminal cytosolic domain termed the recognition complex assembly
unit, and a C-terminal membrane embedded domain, forming the β-barrel type protein
channel (Hinnah et al., 2002; Ertel et al., 2005). The main preprotein receptor of the
TOC complex has been identified as Toc34 (Balsera et al., 2009). Toc34 is anchored to
the membrane by a single transmembrane helix in the C-terminal end of the protein and
contains a large GTPase domain located in the cytosol (May and Soll, 1998; Sun et al.,
2002; Koenig et al., 2008). Based on specific energy requirements, the import of
proteins into plastids can be initially divided into three steps (Perry and Keegstra, 1994;
Kouranov and Schnell, 1997): (1) reversible binding on the plastid surface independent
of nucleotides; (2) stable binding and insertion at < 100 µM ATP in the presence of
Chapter 1 General Introduction
6
GTP (Kessler et al., 1994); and (3) translocation across the outer and inner envelope
requiring > 100 µM ATP (Pain and Blobel, 1987; Theg et al., 1989).
The recognition of plastid precursor proteins by the GTPase receptors on the
outer envelope surface has been intensively studied (Balsera et al., 2009). There are
currently two models for precursor recognition. The first favours Toc159 as the initial
receptor (Hiltbrunner et al., 2001; Bauer et al., 2002; Smith et al., 2004). The process of
GTP hydrolysis and oligomerisation of both GTPases results in the transfer of precursor
proteins to the Toc75 import channel (Hirsch et al., 1994; Schnell et al., 1994; Ma et al.,
1996). Transport through the Toc75 channel and across the membrane is then furthered
by the inter-envelope space located Hsp70 (Perry and Keegstra, 1994). This hypothesis
has been challenged by a second model, which proposes that Toc34 is the initial
receptor for plastid precursor proteins (Sveshnikova et al., 2000; Schleiff et al., 2003;
Becker et al., 2004). Electron microscopy of purified TOC complex suggests that the
TOC complex contains a toroidal structure composed of four protein channels enclosing
a central protruding domain (Schleiff et al., 2003). Stoichiometric analysis of the TOC
complex further identified that it contained four Toc75 proteins, four Toc34 proteins,
and a single Toc159 protein (Schleiff et al., 2003). It has been speculated that the
central Toc159 represents the GTP-driven import motor, which moves preproteins
through the import channel after receiving them from Toc34 (Balsera et al., 2009). A
single Toc159 molecule could alternatively interact with the four Toc34 receptors in a
rotational ‘on and off mode’ (Sveshnikova et al., 2000; Schleiff et al., 2002; Schleiff et
al., 2003). It has also been observed that a TOC complex consisting of Toc159 and
Toc75 can import precursor proteins into liposomes demonstrating a minimal TOC
complex (Balsera et al., 2009). A different study led to the conclusion that the G-
domain of Toc159 was not necessary for protein import (Chen et al., 2000; Lee et al.,
2003, precursor proteins could completely bypass the receptor subunits and bind
directly to Toc75 {Chen, 2000 #161)(Chen et al., 2000). Consequently, import was
achieved without the G-domain of Toc159. Although the two models agree that the
GTP mediated regulation of the Toc34/Toc159 receptors and the central channel
function of Toc75 are both required for protein import, there are still conflicting views
about the exact specifics of recognition and translocation.
Chapter 1 General Introduction
7
1.3.4 Inner envelope import machinery: TIC complex
The composition and stoichiometry of the membrane complex for import into
chloroplasts at the inner envelope membrane is not as well defined as the TOC
machinery. The TIC complex is composed of between 7 and 8 proteins in higher plants,
including Tic110 and Tic20, which form the putative constituents of the translocon
channel, the cochaperone Tic40 and the translocon associated proteins Tic55, Tic32 and
Tic62 (Stengel et al., 2007). The individual and cooperative functions of these proteins
are still unknown. The identity of the most important part of the complex, the translocon
protein conducting channel, still remains elusive. In the case of the TOC translocon,
Toc75 has been unequivocally identified as an essential channel forming protein. In the
case of the TIC complex there is still much discussion as to the identity of the
translocation channel. Several candidates have been put forward to fulfil this essential
role, including Tic110, Tic20, and Tic21 (Chen et al., 2002; Heins et al., 2002; Teng et
al., 2006). Tic21 and Tic20 both show structural similarities to the translocation channel
proteins from the mitochondrial inner membrane Tim17 and Tim23. However,
biochemical evidence for this function is lacking (Rassow et al., 1999; Teng et al.,
2006). Tic20 has been shown to be an essential protein for plant development (Chen et
al., 2002). It has been proposed that Tic20 plays a regulatory role and may also be
involved in TIC complex assembly (van Dooren et al., 2008).In contrast, Tic110
displays features that make it a good candidate for the translocation channel: it is
conserved throughout plastid types across multiple species (Davila-Aponte et al., 2003);
is expressed in cells in comparative amounts to Toc75 (Vojta et al., 2004); shows
channel activity in vitro (Heins et al., 2002); has been found to be associated with
precursor proteins and chaperones (Lubeck et al., 1996; Nielsen et al., 1997); can form
super complexes with the TOC complex (Schnell and Blobel, 1993); and is essential for
plant viability (Inaba et al., 2005). In any case, Tic110 has an essential role in preprotein
recognition on the trans side of the outer membrane and associates with Tic40 to link
the central translocation channel and import motor (Kovacheva et al., 2005).
Apart from the main channel forming components of the motor module, the TIC
complex also contains a number of regulatory members (Balsera et al., 2009). They all
contain features for sensing the redox state of the chloroplasts and thereby regulate
import at the inner envelope, according to the specific requirements of the plastid
(Balsera et al., 2009). It has been known for sometime that at least two precursors; non-
Chapter 1 General Introduction
8
photosynthetic ferredoxin (FdIII) and FNR isoform II (FNRII), are differentially
imported in the light and dark (Hirohashi et al., 2001).
1.3.5 Thylakoid protein import
The thylakoid membrane is a highly specialised membrane that contains the
photosystems and the ATPase complex, which are involved in photosynthetic electron
transfer, coupled to the chemiosomotic process. The abundant photosynthetic machinery
of the thylakoid membrane is composed of subunits encoded by both nuclear and
plastidic genomes, in contrast to all known plastid luminal proteins, which are nuclear
encoded (Balsera et al., 2009). Proteomics and other analyses have identified that
systems homologous to the Sec, Tat, and YidC machineries are found in the thylakoids,
termed the cpSec, cpTat and Alb3 machinery respectively (Schunemann, 2007). These
machineries are responsible for the import and assembly of thylakoidal proteins
(Schunemann, 2007). Some subunits of the thylakoids are targeted by a bipartite
targeting signal at their N-terminus. The first part directs the proteins through the outer
and inner envelopes to the stroma, where the protein is cleaved by the SPP into an
intermediate form of the protein. The second part of the targeting signal guides the
intermediate form of the protein into the thylakoids, where it is processed by the
thylakoidal processing peptidase, thereby generating the mature form of the protein
(Sakamoto, 2006).
1.4 Mitochondrial protein targeting and import
Most research concerning the targeting and import of proteins into mitochondria
originates from studies on the model organism Saccharomyces cerevisiae (yeast)
(Figure 1.2). Import into mitochondria requires several membrane bound complexes:
the translocase of both the outer and inner membranes (TOM and TIM respectively); the
sorting and assembly machinery (SAM) which inserts β-barrel proteins into the outer
membrane; and the presequence translocase associated motor (PAM) which is required
for translocation into the matrix (Figure 1)(Chacinska et al., 2009). There is also another
pathway termed the mitochondria import and assembly (MIA) pathway for the import
of intermembrane space proteins (Chacinska et al., 2004).
1.4.1 Mitochondrial targeting signals
Mitochondrial targeting signals are generally located at the N-terminus and are
between 20 and 60 amino acids in length, are enriched in positively charged and
Chapter 1 General Introduction
9
hydroxylated residues, and have the ability to form an α-amphipathic helix.
Mitochondrial targeting signals are also generally cleaved upon import. Although
sequence conservation at the cleavage site is low, most contain an arginine residue at
position -2 or -3 (Schneider et al., 1998). The mitochondrial targeting signal is crucial
for interaction with the TOM complex on the outer membrane of mitochondria. A large
number of mitochondrial proteins have also been shown to contain internal targeting
signals; the characteristics of which are much less defined than N-terminal targeting
signals (Neupert and Herrmann, 2007). This class of targeting signals is found on all
outer membrane proteins and also a number of proteins destined for the matrix and
inner membrane (Neupert and Herrmann, 2007). Hydrophobic proteins destined for the
inner membrane, including mitochondrial carrier proteins, mainly contain internal
targeting information within their transmembrane domains (Neupert and Herrmann,
2007). All mitochondrial targeting signals are recognized by specific receptors of the
TOM complex and guided to their specific translocons of the inner membrane.
1.4.2 Mitochondrial protein import
TOM complex
With a few exceptions, proteins that are imported into mitochondria must first
interact with the TOM complex. The TOM complex is a 400 kDa protein complex
consisting mainly of the integral membrane protein Tom40, which forms the protein
channel and a number of smaller single α-helical transmembrane subunits. Also
associated with the TOM complex are the primary receptors for mitochondrial targeting
signals, anchored to the membrane by a single transmembrane helix. Tom40 contains a
predicted amphipathic β-barrel structure embedded into the membrane (Hill et al.,
1998). The receptor domains of the TOM complex contain a hydrophobic groove,
which interacts with the hydrophobic signals or the hydrophobic surface of
mitochondrial targeting signals (Abe et al., 2000; Chan et al., 2006). Upon receptor
binding of mitochondrial targeting signals, the protein is transferred to the translocation
pore of Tom40. According to the binding chain hypothesis, the translocation of proteins
through the TOM complex does not rely on ATP or the membrane potential but rather
on an interaction with several binding sites with increasing affinity to cross the outer
membrane (Meisinger et al., 2001).
SAM complex
β-barrel proteins of the mitochondrial outer membrane are inserted by a
specialised integration pathway. After transport through the TOM complex, β-barrel
Chapter 1 General Introduction
10
proteins are recognised by small TIMs in the intermembrane space (IMS) and guided to
the SAM complex. The SAM complex is made up of a number of integral membrane
subunits, the most notable Sam50. Sam50 is a member of the Omp55 family of proteins,
involved in the insertion of outer membrane proteins in bacteria (Paschen et al., 2003).
Sam50, which is conserved in most organisms, is thought to be the integrase subunit of
the SAM complex. The integration of β-barrel proteins into the outer membrane is
independent of ATP and is thought to be an energetically favourable process. However,
the actual mechanistic action of the SAM complex is yet to be determined. Recently the
SAM complex has also been implicated in the insertion of α-helical proteins into the
outer membrane (Stojanovski et al., 2007).
IMS import
All mitochondrial proteins destined for the IMS are nuclear encoded and
generally do not contain classical mitochondrial targeting signals. There are two main
pathways for proteins to be imported into the IMS (Herrmann and Hell, 2005). The first
describes the import of IMS proteins that contain a mitochondrial targeting signal with a
bipartite presequence, which is characterised by a hydrophobic sorting signal
downstream of the targeting signal. The first most N-terminal part of the targeting
signal directs the protein to the TIM23 complex, where the hydrophobic region anchors
the protein into the inner membrane. A processing step in the IMS proteolytically
removes the presequence behind the transmembrane region, leaving behind a soluble
mature IMS protein [In yeast both cytochrome C1 and cytochrome b2 are processed in
this manner (Gasser et al., 1982)]. However, most IMS proteins do not contain a
cleavable N-terminal mitochondrial targeting signal. Following TOM complex
translocation IMS proteins interact with specific factors within the IMS. These factors
promote oxidative folding events which lead to the trapping of the proteins in the IMS.
The IMS of mitochondria contains a complete set of machinery to catalyse the oxidative
folding, and reflects the evolution of these components from the periplasmic space of
bacteria (Mesecke et al., 2005). Substrate proteins, which are specific for the
mitochondrial IMS import and assembly (MIA) pathway contain conserved cysteine
residues. After passage through the TOM complex, IMS destined proteins interact with
the essential protein Mia40, which forms intermolecular disulfide bridges within the
IMS destined protein (Chacinska et al., 2004). Upon release from Mia40, the substrate
proteins are oxidised into functionally folded proteins. For reoxidation, Mia40 must
interact with the sulfhydryl oxidase Erv1 which itself is regenerated by transferring
Chapter 1 General Introduction
11
electrons to cytochrome c and the respiratory chain (Mesecke et al., 2005). The final
release of electrons to molecular oxygen completes the electron transfer chain of the
intermembrane space assembly pathway.
TIM23 and TM22 complexes
There are two distinct pathways for import into the mitochondrial matrix and
insertion into the inner membrane. The general import pathway directs proteins with
mitochondrial targeting signals to the TIM23 complex, whilst the carrier import
pathway inserts proteins into the membrane via the TIM22 complex. Once the precursor
proteins reach the matrix, the presequence is removed by the mitochondrial processing
peptidase and molecular chaperones assist the folding and assembly of precursor
proteins into functional complexes.
TIM23 complex
The TIM23 complex is responsible for the translocation of proteins into the
matrix and for the import of a limited number of inner membrane and IMS proteins. In
all cases, TIM23 substrates contain an N-terminal targeting signal. Inner membrane
destined proteins containing a single transmembrane helix use a stop transfer
mechanism, where the transmembrane helix acts as the stop transfer signal. If an inner
membrane protein contains more than one transmembrane region, it may be fully
imported into the matrix first and then subsequently relocated to the inner membrane by
Oxa1p, which is a YisC/Alb3 homolog (Hell et al., 1998). Oxa1p is also involved in the
insertion of mitochondrially encoded proteins into the inner membrane from the matrix
(Hell et al., 1998; Luirink et al., 2001). In general, proteins are passed from the TOM
complex to the TIM23 complex by interacting with receptor like domains of the TIM23
complex in the IMS (Mokranjac et al., 2003). The membrane potential across the inner
membrane provides the energy source for translocation. Similar to translocation through
the TOM complex, precursors moving through the TIM23 complex interact with a
number of different subunits until fully translocated into the matrix, upon which the
precursor proteins are cleaved and folded into fully functional proteins. During the
import of matrix targeted proteins, the TIM23 complex becomes associated with the
PAM complex. The PAM complex is comprised of a number of small subunits, the
major constituents being mtHsp70 and the nucleotide exchange factor Mge1. The exact
role of the PAM complex is not yet completely understood, although it is thought to
provide two important roles during the import of matrix targeted proteins: the first is the
Chapter 1 General Introduction
12
active pulling of preproteins through the TIM23 channel and the second is the passive
trapping of preproteins within the matrix by binding mtHsp70. The cooperation between
the TIM23 and PAM complex results in the import and correct folding of matrix
targeted proteins.
TIM22
Most inner membrane proteins that are synthesised in the cytosol do not contain
a cleavable targeting signal and characteristically contain an even number of
transmembrane helices with both the N or C-termini oriented to the IMS. These internal
targeting signals direct the protein to the TIM22 complex of the inner membrane for
insertion into the inner membrane. This import pathway is termed the carrier import
pathway due to the initial characterisation using the highly abundant carrier proteins.
This pathway not only includes the membrane bound TIM22 subunits but also requires
the small TIMs from the IMS. The voltage dependent channel forming protein TIM22 is
predicted to contain four transmembrane helices (Kovermann et al., 2002). Import of
TIM22-mediated proteins comprises several steps, which start with initial binding at the
TOM complex on the mitochondrial surface (Rehling et al., 2004). After emerging from
the TOM complex, carrier proteins bind to the small TIMs in the IMS, shielding the
hydrophobic domains from unproductive interactions in the IMS and guiding the
protein to TIM22. The insertion of proteins by TIM22 into the inner membrane is
strictly dependent on the membrane potential. Once carrier proteins reach the
membrane, they are assembled into functional dimers (Neupert and Herrmann, 2007).
1.5 Protein import into plant mitochondria
Although the plant mitochondrial import apparatus displays many similarities
with yeast, significant differences have been observed (Figure 1.3). With reference to
the main translocases, the pore or channel forming subunits; Tom40, Tim22, Tim17,
and Tim23, are highly conserved across all known organisms and the same is true for
plants, with one major exception (Lithgow and Schneider, 2010). The plant Tim17
protein contains an extra C-terminal extension when compared to yeast, which has been
demonstrated to be inserted into the outer membrane (Murcha et al., 2003; Murcha et
al., 2005). The exact role of this extension is still unclear. While the main channel
forming subunits of the translocases are highly conserved, this cannot be said for a
number of other components. When the TOM complex from plants was first purified
and components identified, a number of differences compared to the model organism
Chapter 1 General Introduction
13
yeast was observed (Jansch et al., 1998; Werhahn et al., 2001). Only two proteins from
plants were shown to be related to yeast (Tom40 and Tom7), the rest do not display
significant sequence similarity to the known components of the yeast TOM complex.
One of the major differences between the yeast TOM complex and the plant TOM
complex is in the receptor subunits. Firstly, the proteins identified as the plant Tom20s
showed no sequence similarity to the yeast Tom20 protein (Werhahn et al., 2001). In
fact the plant Tom20 proteins are an elegant case of convergent evolution (Lister and
Whelan, 2006; Perry et al., 2006). Although the yeast and plant proteins are not
evolutionary related to each other they have been shown to have similar tertiary
structures, and in fact contain very similar domains (Perry et al., 2006). The yeast
Tom20 contains a N-terminal transmembrane domain with the receptor domain at the
C-terminus, the plant proteins show the opposite orientation with the transmembrane
domain at the C-terminus and receptor domain at the N-terminus.
In analysing the TOM complex it was discovered that plants do not contain a
Tom22 receptor but contain a protein related to Tom22 of 9 kDa in size, called Tom9
(Werhahn et al., 2001). Further analysis of the plant Tom9 protein showed that contains
a single transmembrane segment similar to Tom22 and a C-terminal trans domain
located in the IMS (Macasev et al., 2004). This trans domain from plants was shown to
have the same function as yeast by complementing a yeast strain deficient in Tom22
(Macasev et al., 2004). Thus the plant Tom9 is the equivalent of yeast Tom22 lacking
the cytosolic receptor domain. One of the most surprising observations about the plant
TOM complex is the absence of a Tom70 like protein (Jansch et al., 1998; Werhahn et
al., 2001). Extensive database and sequence searches failed to identify a Tom70 like
protein in plant genomes. Thus it was of great interest when a Toc64 like protein was
identified on the outer mitochondrial membrane of Arabidopsis (Chew et al., 2004).
Toc64 is a TPR protein found on the outer envelope of chloroplasts and is involved in
chloroplast protein import (Qbadou et al., 2006). It has been hypothesised that this
mitochondrial Toc64 like protein may if fact be a plant Tom70 protein (Chew et al.,
2004). This is due to the similar features of Toc64 and Tom70. Tom70 contains an N-
terminus transmembrane domain followed by 11 TPR segments, which are responsible
for precursor binding and interacting with chaperones during mitochondrial protein
import (Li et al., 2009; Mills et al., 2009). Toc64 also contains an N-terminus
transmembrane domain and contains 3 TPR segments at the C-terminus, which are
thought to be required for precursor binding and also interacting with cytosolic
chaperones (Qbadou et al., 2006). Finally, a unique feature of the plant import apparatus
Chapter 1 General Introduction
14
is the location of the mitochondrial processing peptidases (MPP). Despite the high
sequence similarity of plant and yeast MPP, plant MPP is an integral component of the
cytochrome bc1 complex of the inner membrane, whereas yeast and mammalian MPPs
are located within the matrix (Glaser and Dessi, 1999).
1.6 Peroxisomal protein import
The targeting and subsequent import of peroxisomal proteins can be divided into
four steps: receptor cargo interaction; docking at the peroxisomal membrane;
translocation and cargo release; and finally, receptor recycling back to the cyctosol
(Brown and Baker, 2008).
1.6.1 Receptor cargo interaction and membrane docking
Peroxisomal proteins are synthesised on free polyribosomes in the cytosol
(Lazarow and Fujiki, 1985). Peroxisomal proteins are imported via two conserved
pathways requiring conserved peroxisomal targeting signals (PTS). The major
difference between peroxisomal and mitochondrial or chloroplast targeting is that PTSs
are recognised by soluble receptors in the cytosol, as opposed to membrane bound
receptors. The most common PTS is the PTS1 signal, which is a carboxy terminal
tripeptide motif with the consensus sequence (S/A/C)(K/R/H)(L/M) (Lametschwandtner
et al., 1998). The predominantly cytosolic receptor for PTS1 containing proteins is
Pex5p, which is structurally divided into two domains. The carboxy domain of the
receptor has a high affinity for PTS1 signals and contains a seven TPR motif helix
bundle which forms a ring structure for ligand binding (Gatto et al., 2000; Stanley et
al., 2006). The deduced crystal structure of Pex5p when cocrystalized to a PTS1 peptide
contained two clusters of three TPRs (1-3 and 5-7) enclosing the peptide. The TPR4
hinge region was shown not to be directly involved in PTS1 binding (Klein et al., 2001).
The amino terminal region of Pex5p has some strictly conserved residues and it is
thought that the peroxisomal targeting information is contained within this region of the
protein (Saidowsky et al., 2001; Otera et al., 2002). Recently it has been demonstrated
that a N526K mutation in the carboxy terminus of Pex5p results in conformational
alterations in the amino terminus, which mimic those induced by PTS1 binding
(Carvalho et al., 2007). As the mutation still allows import of Pex5p into peroxisomes
without a bound cargo, it is thought that the triggering mechanism for docking and
translocation into peroxisomes originates from Pex5p.
Chapter 1 General Introduction
15
The second pathway for peroxisomal import involves the peroxisomal targeting
type 2 signal (PTS2) which is located near the N-terminus and consists of the sequence
RLXXXXX(H/Q)L (Lazarow, 2006). While only a small number of proteins utilise this
pathway in yeast, there appear to be many more in plants. This pathway appears to have
been lost in Caenorhabditis elegans (Motley et al., 2000; Reumann et al., 2004). The
cytosolic receptor for PTS2 containing proteins is Pex7p, predicted to contain a seven
bladed β-propellor domain with each blade consisting of a WD40 repeat (Marzioch et
al., 1994; Zhang and Lazarow, 1995). Similar to Pex5p, Pex7p also shuttles between the
cytosol and the peroxisome during cargo translocation (Nair et al., 2004). However,
Pex7p does not work independently, as there are several accessory proteins required for
delivery of PTS2 containing proteins to peroxisomes (Stein et al., 2002). In yeast, there
are two structurally related peroxins, Pex18p and Pex21p, which are crucial for the
import of PTS2 containing proteins (Purdue et al., 1998). In plants, however, the
situation is slightly different as Pex5p and Pex7p form a PTS1/PTS2 receptor complex,
with the amino terminal domain of Pex5p interacting with the carboxy terminal of
Pex7p (Nito et al., 2002). This interaction has been shown experimentally, as a down
regulation of Pex5p results in a PTS2 import defect, suggesting that PTS1 and PTS2
protein import is coupled in plants in a similar manner to that seen in mammals
(Hayashi et al., 2005).
Once the respective PTS receptors Pex5p and Pex7p have bound their correct
cargo, they are then targeted to the peroxisomal membrane surface. At the peroxisomal
membrane surface the receptor cargo complex interacts with a number of membrane
proteins, before being translocated into the peroxisomal matrix (Brown and Baker,
2008).
1.6.2 Receptor cargo translocation, cargo release and receptor recycling
The translocation of the receptor cargo complex into peroxisomes has been
operationally defined as a peroxisome associated protease resistant state (Brown and
Baker, 2008). This can be explained by the two current hypotheses for the translocation
of proteins into the peroxisome: the extended shuttle hypothesis, where the receptor
cargo complex completely enters the peroxisomal matrix; or the simple shuttle
hypothesis, where the receptor cargo complex is embedded into the membrane, with the
cargo released into the matrix and the receptor remaining protease protected in the
membrane (Brown and Baker, 2008).
Chapter 1 General Introduction
16
There has been much debate in the literature over the extended versus simple
shuttle hypotheses, with evidence readily found for both (Rachubinski and Subramani,
1995; Kunau, 2001; Smith and Schnell, 2001). It has been demonstrated that when GFP
is fused to the carboxy terminus of Pex7p, the intracellular distribution shifted from
mainly cytosolic to peroxisomal. When the GFP was subsequently cleaved, the GFP
remained in the peroxisome whereas Pex7p was observed to exit the peroxisome back to
the cytosol (Nair et al., 2004). While it has now been clearly demonstrated that both
Pex5p and Pex7p receptors do enter the peroxisome, it is still unclear whether they
simply remain embedded in the membrane, with their cargo binding site exposed to the
matrix or whether they are fully translocated into the matrix along with their cargo
(Dammai and Subramani, 2001).
In both mammals and plants the PTS2 containing sequence of a peroxisomal
protein is proteolytically removed after import. The removal of the PTS2 sequences is
not tightly linked with import, as both cleaved and uncleaved forms of thiolase have
been observed with in vitro imports into rat liver peroxisomes (Miura et al., 1994). The
enzyme responsible for this cleavage in mammals is termed trypsin domain-containing
domain 1 (TYSND1). A related enzyme in plants, Deg15, has been identified to carry
out the same processing step in Arabidopsis and watermelon (Helm et al., 2007;
Kurochkin et al., 2007).
The exact mechanistic details underlying the translocation and components of
the translocon are still lacking. It has been proposed that the components that make up
the docking complex on the peroxisomal membrane form part of the translocon (Brown
and Baker, 2008). The possible multiple binding sites for the Pex5p receptor on the
peroxisomal membrane have suggested the existence of an import cascade of a cargo
loaded receptor, as it interacts with different components of the import machinery
(Baker and Sparkes, 2005). It has been observed that Pex5p changes its characteristics
when it is associated with the peroxisomal membrane, as it behaves as an integral
membrane protein when it interacts with the docking complex (Gouveia et al., 2000).
Taken together with the observation that Pex5p can spontaneously insert into lipid
membranes, this suggests that a population of Pex5p receptors actually form the import
pore via protein lipid interactions, leading to an opening of the membrane allowing the
entry of a second cargo loaded Pex5p (Erdmann and Schliebs, 2005; Kerssen et al.,
Chapter 1 General Introduction
17
2006). This hypothesis is referred to as the transient pore model (Erdmann and Schliebs,
2005).
Once the loaded cargo receptor complex enters the peroxisome the cargo must
be released into the matrix by the receptor, although little is known about the exact
mechanism of the process. In vitro experiments have indicated a displacement model for
receptor release, where a protein actively displaces the loaded cargo from the receptor
(Agne et al., 2003). Once the cargo is unloaded, both Pex5p and Pex7p can return to the
cytosol and take part in further rounds of import (Baker and Sparkes, 2005). The
dislocation and recycling of receptors from the peroxisome requires the action of the
receptor recycling complex, the mechanism for which is not yet fully understood (Agne
et al., 2003).
1.7 Dual targeting
The traditional dogma of molecular biology is that one gene gives rise to one
protein, which subsequently has one location. However, this no longer appears valid in
post-genomic biology. It has become clear with the sequencing of a number of
genomes, that the complexity of the proteome exceeds that of the genome in terms of
functional units, (i.e., there are more proteins than genes). This observed complexity
could be achieved in a number of different ways. Alternative splicing and protein
modifications are the best characterised processes to date (Kazan, 2003; Siuti and
Kelleher, 2007; Witze et al., 2007). Another mechanism that can increase the
complexity of proteomes is transcript editing of both nuclear and organellor genomes
(Nishikura, 2006; Takenaka et al., 2008). The dual targeting of proteins does not
increase the number of proteins, but it can expand the function(s) of a protein located in
two or more locations, because presumably, it functions in a distinct biochemical
process at each different location. A dual targeted protein is defined as the product(s) of
one gene targeted to two or more locations and was first characterised in 1995 for the
Pea glutathione reductase (GR), which was reported to be targeted to both chloroplasts
and mitochondria (Creissen et al., 1995). The number of identified dual targeted
proteins represents only a small proportion of the organeller proteomes. However, the
small number of characterised dual targeted proteins may only represent the tip of the
iceberg. While the majority of dual targeted proteins in plants are targeted to
mitochondria and chloroplasts, there are many other examples of dual targeting,
including mitochondria, plastids, and cytosol (Small et al., 1998), mitochondria and ER
Chapter 1 General Introduction
18
(Bhagwat et al., 1999), mitochondria and the nucleus (Krause and Krupinska, 2009),
peroxisomes and mitochondria (Petrova et al., 2004), plastids and the cytosol (Kiessling
et al., 2004) and mitochondria and the cytosol (Regev-Rudzki et al., 2005). With the
amount of knowledge being gained from complete genome sequencing, combined with
the emerging information from organelle proteomic studies, GFP studies and prediction
programs, the number of dual targeted proteins has been increasing steadily over the
past 15 years since their discovery (Cho et al., 1999; Koroleva et al., 2005; Heazlewood
et al., 2007). Much work has been carried out to understand the mechanisms involved in
dual targeting. In particular alternative transcriptional initiation or splicing and
ambiguous targeting signals have been investigated (Peeters and Small, 2001; Karniely
and Pines, 2005). Alternative transcriptional initiation or splicing represent
transcriptional or post transcriptional events, which produce proteins translated with two
different targeting signals (Dinkins et al., 2008). Ambiguous targeting signals target a
protein to two locations, with the signals being indistinguishable from each other.
1.7.1 Ambiguous targeting signals
As discussed previously, mitochondria and chloroplasts have separate and
distinct targeting signals that can mediate their targeting and import into each organelle.
However, a small subset of these proteins contain ambiguous targeting signals that
target proteins to both organelles. This definition applies to the product of a single gene,
which gives rise to one protein, which is then targeted and imported into both
mitochondria and chloroplasts (Peeters and Small, 2001). Since the discovery of the
first ambiguously dual targeted protein, GR, a range of different proteins from various
biosynthetic pathways (transcription, translation and protein degradation) have been
demonstrated to be dual targeted by ambiguous targeting signals (Peeters and Small,
2001; Elo et al., 2003; Silva-Filho, 2003).
Analysis of ambiguous targeting signals has shown that they are similar to both
chloroplast and mitochondrial targeting signals, in that they are enriched in positively
charged residues and deficient in acidic residues such as glycine (Pujol et al., 2007).
However, there are no known distinguishing features that can separate ambiguous dual
targeting signals from mitochondrial and/or chloroplastidic specific targeting signals.
They appear to fall somewhere in between mitochondrial and chloroplast targeting
signals in their content of serine and arginine residues and are possibly slightly enriched
in hydrophobic residues. It has been shown that, in yeast, for a protein targeted to the
mitochondria and another location, its mitochondrial targeting signal is weaker
Chapter 1 General Introduction
19
compared to mitochondrial proteins determined using the MITOPROT prediction
program. However no such evidence has been found in plants (Claros and Vincens,
1996; Dinur-Mills et al., 2008).
To date the most characterised ambiguous dual targeted signal has been that of
the Pea GR (Rudhe et al., 2002; Chew et al., 2003; Rudhe et al., 2004). Studies
involving deletion and site directed mutagenesis have revealed that some regions in the
targeting signal are more important for targeting to one organelle, but overall the dual
targeting signal overlaps (Chew et al., 2003). This study is consistent with other studies
carried out on tandem arrangements of mitochondrial and chloroplast targeting signals,
which demonstrated that passenger proteins are targeted by the most N-terminal signal
(de Castro Silva Filho et al., 1996). In the case of GR, it was found that positive
residues throughout the signal and hydrophobic residues at the N-terminus affected the
import into mitochondria whilst the hydrophobic residues had the greatest affect on
chloroplast import (Chew et al., 2003). In addition, it has also been observed that
arginine plays an important role in the mitochondrial import of three dual targeted
tRNA synthetases (Pujol et al., 2007). A recent study into dual targeting signals
concluded that while there is no general rule for the determinants of dual targeting, the
N-terminal portion is essential for the import into both mitochondria and chloroplast
(Berglund et al., 2009).
1.7.2 Alternative mechanisms of dual targeting
Post-translational mechanisms that result in the dual targeting of a protein are
usually found in non-plant organisms. In yeast, two enzymes of the TCA cycle,
fumarase and aconitase, have both been shown to be distributed between the cytosol and
mitochondria (Karniely and Pines, 2005). The mechanism for this dual distribution
involves the reverse translocation of a subset of molecules back into the cytosol
(Karniely and Pines, 2005). While the cytosolic presence of fumarase is quite obvious
(50% of the total fumarase is located in the cytosol), the amount of aconitase in the
cytosol is very small (less than 5%) (Sass et al., 2003; Regev-Rudzki et al., 2005).
Recently the abundance of fumarase in the cytosol compared to the mitochondria was
shown to be controlled by intracellular metabolite clues (Regev-Rudzki et al., 2009).
More specifically, it was suggested that metabolites from the glyoxylate shunt can act as
nanosensors for fumarase distribution (Regev-Rudzki et al., 2009), showing a complex
mechanism of control. Whilst no such pathway has been identified in plants, external
Chapter 1 General Introduction
20
factors such as light and stress have been suggested to influence dual targeting (Silva-
Filho, 2003).
A single gene may also be alternatively transcribed from two different exons or
alternatively spliced to produce two separate messages encoding proteins targeted to
different locations (Peeters and Small, 2001). Some genes use multiple translation start
sites to determine dual targeting, for example, the longer protein is targeted to one
organelle and the shorter protein targeted to a second organelle (Chabregas et al., 2001;
Kobayashi et al., 2001; Watanabe et al., 2001; Hedtke et al., 2002). This example has
been reported in Arabidopsis with DNA polymerase γ2, which is dual targeted via the
use of a non AUG start codon (CUG), which adds an additional seven amino acids to
the N-terminus (Christensen et al., 2005). When translation starts at the standard AUG,
the protein is targeted to plastids, but when translation starts at the alternative CUG site,
the protein is targeted to both mitochondria and plastids (Christensen et al., 2005).
1.7.3 Systematic studies of dual targeted proteins
Studies that have investigated the biochemical processes common to both
mitochondria and chloroplasts have identified a number of dual targeted proteins. The
ascorbate glutathione cycle of Arabidopsis was originally thought to be housed solely in
chloroplasts, to remove the large amounts of H2O2 generated by photosynthetic
reactions. However biochemical studies have also measured the activity of ascorbate
glutathione cycle enzymes in the mitochondria of various plants. A study demonstrated
that the enzymes involved in the ascorbate glutathione cycle, (ascorbate peroxidase
(APX), monodehydroascorbate reductase (MDHAR) and GR) were in fact dual targeted
to both mitochondria and chloroplasts in Arabidopsis (Chew et al., 2003). This was the
first evidence for proteins of an entire biochemical pathway to be targeted to both
mitochondria and chloroplasts.
A study involving organellor tRNA synthetases has also demonstrated that most
organellor tRNA synthetases are dual targeted to mitochondria and chloroplasts
(Duchene et al., 2005). This is not surprising, as both mitochondria and chloroplasts
contain their own genome, which must be replicated, transcribed and translated. It has
also been suggested that most of the proteins involved in organelle DNA and RNA
metabolism are in fact dual targeted (Elo et al., 2003).
Chapter 1 General Introduction
21
These previous studies have identified dual targeted proteins by focusing on the
targeting ability and/or location of single gene product(s) or small gene families. So far
there has been no genome-wide search for dual targeted proteins in Arabidopsis.
Therefore, it is possible that there are many more dual targeted proteins in Arabidopsis
that have yet to be identified. It was hypothesised that a global approach to the analysis
of protein locations may assist in the identification of additional dual targeted proteins.
The overall aim of this research was therefore to employ a systematic and global
approach to the identification of dual targeted proteins. This was attempted in order to
determine the extent of dual targeting of proteins in plants, with particular emphasis on
the dual targeting of proteins to the mitochondria and another location. A further aim of
this research was to investigate the mechanisms (i.e., receptors and signals) involved in
the dual targeting of proteins to mitochondria.
1.8 Research proposal
The specific aims of this PhD study are:
1. To identify proteins present in multiple organelles in plants (plastids,
mitochondria and/or peroxisomes).
2. To investigate the mechanism of dual targeting by identifying the import
receptors and machinery responsible for import of dual targeted proteins to the
mitochondria.
To achieve these aims, it was necessary to identify proteins present in multiple
organelles. This list of proteins was generated using bioinformatic resources available
from previous studies. First, a list of proteins experimentally defined as located in
mitochondria was compiled using the SUBA database (Heazlewood et al., 2007), and a
list of proteins predicted to target to peroxisomes was compiled using the Araperox
database (Reumann et al., 2004). These lists were cross-referenced to form a list of
candidate dual targeted proteins for mitochondria and peroxisomes. Second, the protein
prediction program, Predotar was used in two different modes (animal only and plant
only) to generate a ranked list of proteins targeted to mitochondria and plastids (Small
et al., 2004). Third, a list of proteins defined experimentally by proteomic approaches as
being present in two or more locations was compiled using the SUBA database
(Heazlewood et al., 2007). Finally a list of candidate dual targeted proteins likely to be
located in both mitochondria and chloroplasts was also compiled (i.e., proteins involved
in DNA transcription and replication).
Chapter 1 General Introduction
22
The compiled lists were then merged into a single list of candidate dual targeted
proteins. Dual targeting of proteins was initially tested using GFP tagging. Results for
selected dual targeted proteins were subsequently confirmed using Western blotting,
mass spectrometry, or in vitro protein import assays.
To gain further insights into the mechanisms of dual targeting, a putative
receptor protein likely to be involved in dual targeting was tested. OM64, a protein
previously identified as located on the outer mitochondrial membrane, shares 70%
amino acids sequence identity with Toc64, a protein that has been proposed to act as a
receptor for plastid protein import (Chew et al., 2004). The functional role of OM64 in
the import of dual targeted proteins into plant mitochondria was analysed in variety of
assays.
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Chapter 2 Subcellular localisation of Arabidopsis thiolases
26
Chapter 2
Subcellular localisation of Arabidopsis thiolases
Chapter 2 Subcellular localisation of Arabidopsis thiolases
27
Foreword to Study I Knowledge of a proteins subcellular localisation is critical in understanding its
function within plant metabolism and the biochemical pathways its involved in. Nearly
a decade ago, it was proposed that the β-oxidation of fatty acids may take place within
the mitochondria of plants (Masterson and Wood, 2001). However, it has also been
proposed that β-oxidation occurs within the peroxisomes of plant cells (Graham and
Eastmond, 2002). A mitochondrial and peroxisomal location for the β-oxidation
pathway in plants is supported by the finding of 3-ketoacyl-CoA thiolase 2 (KAT2)
activity within both organelles (Footitt et al., 2002). KAT2 is the terminal enzyme in the
β-oxidation pathway in plants. In Arabidopsis KAT2, has been identified as a
mitochondrial protein in proteomic studies using purified mitochondria (Kruft et al.,
2001),(Heazlewood et al., 2004). Continuing from these, another study proposed a role
for KAT2 in isoleucine catabolism in plant mitochondria (Taylor et al., 2004). KAT2 is
predicted by three different subcellular prediction programs to be mitochondrial
(Heazlewood et al., 2005), however its protein sequence has also been shown to contain
a PTS2 sequence within the N-terminal part of the protein (Baker and Sparkes, 2005).
Based on prediction, proteomic, and activity analysis, KAT2 seemed a likely
candidate for a dual targeted protein between mitochondria and peroxisomes. Study I
aimed to clarify the subcellular localisation of KAT2 and define the localisation of any
other known thiolase like proteins in Arabidopsis. This was achieved by identifying all
thiolase like genes from Arabidopsis, and performing in vitro and in vivo organelle
targeting assays to define their subcellular localisations. It was found that the
Arabidopsis genome contains three genes encoding 3-ketoacyl-CoA thiolases (KAT),
one of which encodes two proteins with different N-terminal sequences. The
Arabidopsis genome also contains two genes encoding acetoacetyl-CoA thiolases
(ACAT), producing a total of five different proteins. In vitro and in vivo organelle
targeting studies identified that 3 KATs and 1 ACAT were peroxisomal, 1 KAT and 4
ACATs were cytosolic, and none were found to be mitochondrial. Its was concluded
that β-oxidation of fatty acids in Arabidopsis does not take place within mitochondria
and the final steps of isoleucine catabolism are most likely carried out in the cytosol.
Abstract The sub-cellular location of enzymes of
fatty acid b-oxidation in plants is controversial. In the
current debate the role and location of particular
thiolases in fatty acid degradation, fatty acid synthesis
and isoleucine degradation are important. The aim of
this research was to determine the sub-cellular location
and hence provide information about possible func-
tions of all the putative 3-ketoacyl-CoA thiolases
(KAT) and acetoacetyl-CoA thiolases (ACAT) in
Arabidopsis. Arabidopsis has three genes predicted to
encode KATs, one of which encodes two polypeptides
that differ at the N-terminal end. Expression in
Arabidopsis cells of cDNAs encoding each of these
KATs fused to green fluorescent protein (GFP) at their
C-termini showed that three are targeted to peroxi-
somes while the fourth is apparently cytosolic. The four
KATs are also predicted to have mitochondrial tar-
geting sequences, but purified mitochondria were un-
able to import any of the proteins in vitro. Arabidopsis
also has two genes encoding a total of five different
putative ACATs. One isoform is targeted to peroxi-
somes as a fusion with GFP, while the others display
no targeting in vivo as GFP fusions, or import into
isolated mitochondria. Analysis of gene co-expression
clusters in Arabidopsis suggests a role for peroxisomal
KAT2 in b-oxidation, while KAT5 co-expresses with
genes of the flavonoid biosynthesis pathway and
cytosolic ACAT2 clearly co-expresses with genes of
the cytosolic mevalonate biosynthesis pathway. We
conclude that KATs and ACATs are present in the
cytosol and peroxisome, but are not found in
mitochondria. The implications for fatty acid
b-oxidation and for isoleucine degradation in
mitochondria are discussed.
Keywords Thiolase Æ Mitochondria Æ Peroxisomes Æb-oxidation Æ Sub-cellular localization
Abbreviations
AOX Alternative oxidase
ACAT Acetoacetyl-CoA thiolase
KAT 3-Ketoacyl-CoA thiolase
Rubisco SSU Small subunit of Ribulose 1,5-
bisphosphate carboxylase/oxygenase
Introduction
It is widely accepted that the complete b-oxidation of
medium- and long-chain fatty acids in plants takes
place in the peroxisomes (Hooks 2002), as it does in
yeast (van Roermund et al. 2003). However, some
biochemical evidence suggests that plant mitochondria
can also carry out such b-oxidation of fatty acids
(Masterson and Wood 2001). It has also become clear
recently that plant mitochondria catalyse at least the
initial steps in the degradation of branched-chain
a-keto acids, derived from leucine, isoleucine and
Electronic supplementary material Supplementary materialis available in the online version of this article at http://dx.doi.org/10.1007/s11103-006-9075-1 and is accessible forauthorized users.
C. Carrie Æ M. W. Murcha Æ A. H. Millar Æ S. M. Smith ÆJ. Whelan (&)ARC Centre of Excellence in Plant Energy Biology,University of Western Australia, MCS building M316,35 Stirling Highway, Crawley 6009 WA, Australiae-mail: [email protected]
Plant Mol Biol (2007) 63:97–108
DOI 10.1007/s11103-006-9075-1
123
Nine 3-ketoacyl-CoA thiolases (KATs) and acetoacetyl-CoAthiolases (ACATs) encoded by five genes in Arabidopsisthaliana are targeted either to peroxisomes or cytosolbut not to mitochondria
Chris Carrie Æ Monika W. Murcha Æ A. Harvey Millar ÆSteven M. Smith Æ James Whelan
Received: 25 June 2006 / Accepted: 10 August 2006 / Published online: 21 November 2006� Springer Science+Business Media B.V. 2006
valine, through a branched chain a-keto acid dehy-
drogenase complex similar to the pyruvate and 2-oxo-
glutarate dehydrogenase complexes of the TCA cycle
(Fujiki et al. 2000; Graham and Eastmond 2002; Taylor
et al. 2004). In the case of the leucine carbon skeleton,
the later steps of degradation are carried out entirely
within the mitochondrion (Graham and Eastmond
2002; Taylor et al. 2004). In contrast, final degradation
of valine derivatives requires both mitochondrial and
peroxisomal steps (Lange et al. 2004). Meanwhile the
complete oxidation of the products from the isoleucine
carbon skeleton includes a b-oxidation step that
requires a 3-ketoacyl-CoA thiolase (KAT) for the
removal of an acetyl-CoA from 2-methylaceto-acetyl
CoA to form propionyl-CoA. However, it is unclear if
this thiolase catalysed b-oxidation of 2-methylaceto-
acetyl CoA occurs in the mitochondrion in plants, as it
does in mammals (Fukao et al. 2001), or whether it
occurs in the peroxisome in plants akin to the final
steps of valine metabolism (Lange et al. 2004).
Comparisons to Saccharomyces cerevisiae are not
informative as yeast degrades branched chain amino
acids not via the branched chain dehydrogenase com-
plex route in mitochondria, but via the Erhlich path-
way involving pyruvate decarboxylase to form the
corresponding aldehydes and an aldehyde dehydroge-
nase to form the corresponding alcohol in the cytosol
(Derrick and Large 1993). Thus yeast does not need a
thiolase for isoleucine degradation.
Two distinct forms of 3-ketoacyl-CoA thiolase are
known. Type 1 3-ketaoacyl-CoA thiolase (KAT; EC
2.3.1.16) is typically involved in the degradative pro-
cess of fatty acid b-oxidation. The Type II enzyme is an
acetoacetyl-CoA thiolase (ACAT; EC 2.3.1.9), typi-
cally involved in the mevalonate pathway where it
functions in the biosynthetic direction. However,
ACATs are not exclusively involved in mevalonate
synthesis. In mammals that undertake both fatty acid
b-oxidation and isoleucine catabolism in mitochondria,
the former is performed by a KAT while the later is
performed by an ACAT (Pereto et al. 2005).
In Arabidopsis, thiolase has been reported to be
associated with both peroxisomes and mitochondria in
sucrose density gradients (Footitt et al. 2002). Kruft
et al (2001) and Heazelwood et al (2004) have both
claimed the thiolase KAT2 encoded by At2g33150 to
be present in purified mitochondria in large-scale
proteome analyses. This thiolase has previously been
proposed to be a component of isoleucine catabolism
in mitochondria (Taylor et al. 2004). However, the
thiolase is question is a type I enzyme, while in mam-
mals it is the mitochondrial type II ACATs that have
been implicated in isoleucine catabolism (Pereto et al.
2005). The KAT2 thiolase encoded by At2g33150 has a
predicted type 2 peroxisomal targeting sequence
(PTS2) conforming to the consensus R-(X)6-H/Q-A/L/F
with a downstream cysteine residue required for pro-
teolytic cleavage (Baker and Sparkes 2005), and is
imported into peroxisomes in vitro (Johnson and
Olsen 2003). At2g33150 is well known to be essential
for peroxisomal b-oxidation (Germain et al. 2001).
However, the protein encoded by At2g33150 is also
predicted to be targeted to mitochondria by three dif-
ferent targeting prediction programs (Heazlewood
et al. 2004). Furthermore, changing a single amino acid
in the peroxisomal targeting signal of the KAT
precursor in mammals, a glutamic acid residue to any
non-acidic residue, resulted in targeting to both mito-
chondria and peroxisomes (Tsukamoto et al. 1994).
This raises the possibility that the thiolase encoded by
At2g33150 is targeted to peroxisomes and mitochon-
dria, representing a dual targeted protein.
This study was carried out to define the subcellular
localization of the products from the putative KATs
and ACATs in Arabidopsis. This was achieved by
identifying all possible thiolase genes in Arabidopsis
and comparing their sequences to known-location type
I and type II thiolases in yeast, mammals and fungi. We
then used transcript sequence data to produce all the
possible cDNAs for these gene products. These
cDNAs were used with in vivo and in vitro organelle
targeting assays to define subcellular localization of
type I and II thiolases in Arabidopsis and gene
expression profiling data was compared to define likely
functional links.
Materials and methods
Identification of genes and cDNAs encoding
thiolase
The predicted protein sequence encoded by At2g33150
previously shown to be a thiolase was used to define
other thiolase encoding genes in Arabidopsis (Germain
et al. 2001), and the sequences from other species as
previously published (Pereto et al. 2005). A similarity
tree was made using ClustalW multiple sequence
alignment and neighbour joining (Thompson et al.
1994, 1997). The gene structures were obtained from
The Arabidopsis Information Resource annotation
version 6 (TAIR6) and three individual cDNAs were
amplified for all possible cDNAs. The cDNAs pro-
duced from the genes were defined using the Arabid-
opsis genome tiling array (Mockler et al. 2005;
Yamada et al. 2003). Targeting predictions of the
98 Plant Mol Biol (2007) 63:97–108
123
encoded proteins were carried out using a variety of
prediction programs: TargetP (Emanuelsson et al.
2000), Mitoprot (Claros and Vincens 1996), Subloc
(Hua and Sun 2001), Ipsort (Bannai et al. 2002),
Predotar (Small et al. 2004), Mitpred (Kumar et al.
2006) and PeroxP (Emanuelsson et al. 2003). Percent-
age identity and similarity was calculated using Mat-
GAP v2.02 (Campanella et al. 2003).
Subcellular targeting of predicted thiolase proteins
The coding sequences of the predicted thiolase pro-
teins were cloned in frame with the coding region of
GFP in pGEM 3Zf(+) containing the 35S CaMV pro-
moter (Chew et al. 2003). The alternative oxidase
(AOX) coding region fused to GFP (Lee and Whelan
2004), and the red fluorescent protein (RFP) fused to a
type 1 peroxisomal SRL targeting signal from pumpkin
(Pracharoenwattana et al. 2005), were used as mito-
chondrial and peroxisomal controls respectively. The
constructs were used to transform Arabidopsis
suspension culture cells by biolistic transformation as
previously outlined (Thirkettle-Watts et al. 2003).
Fluorescence patterns were obtained 48 h after trans-
formation by visualization under an Olympus BX61
fluorescence microscope and imaged using the CellR
imaging software. In vitro protein import assays into
mitochondria isolated from Arabidopsis suspension
cell cultures were carried out as described in Lister
et al. (2004). In vitro mitochondrial uptake assays were
performed by adding precursor protein to 100 lg of
isolated mitochondria in 200 ll in the presence of
respiratory substrate (succinate 5 mM) and ATP
(1 mM) and ADP (200 lM) in import buffer (0.3 M
sucrose, 50 mM KCl, 10 mM MOPS pH 7.2, 5 mM
KH2PO4, 1% (w/v) BSA, 1 mM MgCl2, 1 mM methi-
onine and 5 mM DTT). Reactions were incubated at
24�C for 20 min then divided into two equal aliquots
and placed on ice. To one aliquot Proteinase K was
added to a final concentration of 40 lg/ml and incu-
bated for 15 min on ice, followed by the addition of
PMSF to 2 mM to terminate protease digestion. The
mitochondria were pelleted, washed twice in ice-cold
import buffer. The final pellet was resuspended in
SDS-PAGE sample buffer and proteins separated in
12% (w/v) polyacrylamide gels, then dried. Radiola-
belled proteins were visualized by exposing to a BAS
TR2040 imaging plate for 24 h and reading on a BAS
2500 Bio imaging analyser (Fuji, Tokyo). Outer
membrane ruptured mitochondria (Mit-OM) were
prepared after the import assay to test for the intra-
organelle location of imported protein. Rupture of the
outer membrane allowed access of added protease to
intermembrane space components or inner membrane
proteins exposed to the intermembrane space. Mit-OM
were prepared by resuspending 100 lg of mitochon-
drial protein in 10 ml SEH buffer (250 mM sucrose,
1 mM EDTA, 10 mM Hepes pH 7.4) and then adding
155 ll of 20 mM Hepes pH 7.4 and incubating on ice
for 20 min. To restore osmolarity 25 ll of 2 M sucrose
and 10 ll of 3 M KCl was added and mixed, re-pelleted
and washed in import buffer. Valinomycin was added
to a final concentration of 1 lM where indicated prior
to the addition of the precursor protein to mitochon-
dria and commencement of the import assay. AOX was
used as a positive control and the small subunit of
Ribulose 1, 5-bisphosphate carboxylase/oxygenase
(Rubisco SSU) as a negative control. As some pre-
cursor proteins displayed protease insensitivity even in
the presence of valinomycin the sensitivity of the pre-
cursor proteins alone to added protease was tested.
Sensitivity of the in vitro synthesized radiolabelled
proteins was tested by adding proteinase K to the
synthesized protein alone in the absence of mitochon-
dria to ensure that the added protease could digest the
protein.
In silico expression analysis
Expression correlation for genes encoding KATs and
ACATs was performed using the Expression Angler
program on the Botany Array Resource (Toufighi
et al. 2005). The Genevestigator Arabidopsis micro-
array database was used to analyse the response of the
genes of interest in this study in a variety of tissue types
(Zimmermann et al. 2004). The meta-analyser tool was
the function utilized, ATH1 22k array wild type only
arrays were chosen and the genes of interest were
selected. The data was visualized using a linear scale
from a total of 1860 array experiments. TMeV (TIGR
Multiple Experiment Viewer) programme was used to
cluster the genes and stresses, and Euclidean distance
and complete linkage were chosen for the hierarchal
clustering (Eisen et al. 1998; Saeed et al. 2003).
Results
The Arabidopsis thiolase gene families
Searches of the Arabidopsis genome identify five loci
with sequence similarity to genes encoding known
thiolase proteins (Germain et al. 2001). Comparison of
predicted amino acid sequences shows that they fall
into two classes. Three loci encode the Type I class of
enzyme, KAT 1, 2 and 5, typically involved in
Plant Mol Biol (2007) 63:97–108 99
123
acetyl-CoA formation in fatty acid b-oxidation by
removal of a 2-carbon chain from a 3-ketoacyl-CoA.
The other two genes encode the type II class of
enzyme, ACAT 1 and 2, typically involved in aceto-
acetyl-CoA formation from two molecules of acetyl-
CoA (Fig. 1A, Supplementary Fig. 1A). The type I
genes have previously been annotated as KAT1, KAT2
and KAT5 (3-ketoacyl-CoA thiolase) (Germain et al.
2001), based on the chromosome on which they are
found (At1g04710, At2g33150 and At5g48880, respec-
tively). All three are closely related to known peroxi-
somal located type I thiolases in human, mouse and
yeast and this cluster also contains representatives from
the fungus Neurospora crassa and the monocot rice
(Oryza sativa). The matrix and membrane-bound type I
mitochondrial thiolases involved in fatty acid degrada-
tion in human, mouse and Drosophila cluster separately
and do not contain members from the completed
genome sequences of fungi, Arabidopsis or rice.
The two Arabidopsis type II thiolases (here referred
to as ACAT1 and ACAT2, At5g47720 and At5g48230,
respectively) cluster with the known cytosolic type II
thiolases from yeast and the cytosolic and mitochon-
drial type II thiolases from human, mouse and Dro-
sophila. The monocot rice also has two type II thiolases
that cluster in this set and N. crassa contains a single
type II gene that clusters with the yeast cytosolic type
II protein.
The sequence divergence of mitochondrial type I
thiolases in mammals from the peroxisomal type I
KATs in plants, fungi and animals makes the presence
of KAT2 in Arabidopsis mitochondria appear unlikely
based on phylogenetic evidence if thiolase location is
conserved. However, the sequence analysis does not
define the location of the type II ACAT proteins in
Arabidopsis and leaves open the possibility of a mito-
chondrial location of at least one of these proteins,
especially given the presence of multiple type II thio-
lases in both plants and mammals.
Definition of the number of products from each
Arabidopsis thiolase gene
Analysis of EST sequences and tiling array data shows
that KAT1 and KAT2 loci each encode single poly-
peptide sequences (Fig. 1B) (Mockler et al. 2005;
Yamada et al. 2003). In contrast, KAT5 encodes two
proteins that differ at the N-terminus due to alternative
RNA splicing that generates either 13 (KAT5.1) or 14
(KAT5.2) exons (Fig. 1B). The N-terminal sequences
of proteins encoded by KAT1, KAT2 and KAT5.2
include putative PTS2-type sequences, while the
protein encoded by KAT5.1 does not (Fig. 1B).
Interestingly, proteins encoded by KAT1, KAT2,
KAT5.1 and KAT5.2 proteins are predicted to be tar-
geted to mitochondria by at least two of three different
programs (Table 1). Analysis of EST sequences and
tiling array data shows that ACAT1 and ACAT2 loci
also encode three and two proteins respectively
(Fig. 1B). Differential RNA splicing results in the
protein encoded by ACAT1.1 lacking ten amino acids
at the C-terminus relative to the protein encoded by
ACAT1.2. The protein ACAT1.3 has 20 different
amino acid residues at the N-terminus relative to
ACAT1.1. None of the proteins has predicted orga-
nelle-targeting information (Table 1). Differential
RNA splicing also accounts for the N-terminal 6 amino
acid residues of the protein encoded by ACAT2.1
being replaced by 11 different amino acid residues in
the case of the protein encoded by ACAT2.2 (Fig. 1B).
Neither protein has predicted organelle-targeting
information (Table 1).
Targeting of type I and II thiolases in vivo
To localize thiolases in vivo, GFP and RFP fusions
were employed. GFP and RFP have been used exten-
sively to study protein targeting to mitochondria,
peroxisomes and chloroplasts (Heazlewood et al.
2005). To demonstrate specific mitochondrial and
peroxisomal targeting in vivo and our ability to
distinguish the two, an AOX–GFP construct (Lee and
Whelan 2004) and an RFP–PTS1 construct (Pracha-
roenwattana et al. 2005), were employed. The two
gene constructs were co-delivered into Arabidopsis
suspension culture cells using a biolistic gene gun
(Thirkettle-Watts et al. 2003). After 48 h individual
cells expressing both GFP and RFP fluorescence were
imaged. The results show that GFP and RFP were
targeted to discrete organelles consistent with specific
targeting to mitochondria and peroxisomes respec-
tively (Fig. 2).
To examine thiolase targeting we made translational
fusions with GFP at the C-terminus since peroxisomal
(PTS2) and mitochondrial targeting sequences are both
N-terminal. Thiolase cDNAs encoding all nine pro-
teins were linked to the GFP coding region and cloned
downstream of the CaMV 35S promoter. They were
Fig. 1 Classification of thiolase (KAT and ACAT) genes andgene structure in Arabidopsis. (A) A phylogenetic tree wasgenerated using the neighbour joining method, using ClustalW ofthiolase proteins from a variety of organisms. KAT = 3-ketoacyl-CoA thiolases, ACAT = acetoacetly-CoA thiolases. (B) Genestructure and predicted proteins encoded by thiolase genes. Thedifferences in the proteins encoded by each locus are indicated inbold where evidence for more than one cDNA exists. The openwhite boxes indicate exons
c
100 Plant Mol Biol (2007) 63:97–108
123
MEKATERQRI LLRHLQPSSS SDASLSASAC LSKDSAAYQY
MEKAIERQRV LLEHLRPSSS SSHNYEASLS ASACLAGDSA
MAPPVSDDSL QPRDVCVVGV ARTPIGDFLG SLSSLTATRL
MNVDESDVCI VGVARTPMGG FLGSLSSLPA TKLGSLAIAA
MAHTSESVNP RDVCIVGVAR TPMGGFLGSL SSLPATKLGS
KKGKYGVASI CNGGGGASAL VLEFMSEKTI GYSAL
MERAMERQKI LLRHLNPVSS SNSSLKHEPS LLSPVNCVSE
MAAFGDDIVI VAAYRTAICK ARRGGFKDTL PDDLLASVLK
KAT1At1g04710
KAT2At2g33150
At5g47720.1
At5g47720.2
At5g48230.1
At5g48230.2
KAT5.1 At5g48880.1
KAT5.2At5g48880.2
ACAT1.1
ACAT 1.2
ACAT 2.2
ACAT 2.1
ACAT 1.3
At5g47720.3
MYLSFDPAVM ATYSSVPVCA DVCVVGVART PIGDFLGSLS
A
B
Plant Mol Biol (2007) 63:97–108 101
123
each delivered into Arabidopsis suspension culture
cells together with the RFP–PTS1 construct. After 48 h
individual cells expressing both GFP and RFP fluo-
rescence were imaged, and the images merged. The
results show that KAT1, KAT2 and KAT5.2 and
ACAT 1.3 were targeted to peroxisomes, as indicated
by coincidence of GFP and RFP images (Fig. 2). In
contrast, ACAT1.1, ACAT1.2, ACAT2.1, ACAT2.2
and KAT5.1 show diffuse fluorescence throughout the
cell indicating that no specific targeting to any orga-
nelle has occurred, suggesting a cytosolic localization.
With peroxisomal targeting of KAT1, KAT2, KAT5.2
and ACAT 1.3, although it was apparent that the
patterns of GFP and RFP were essentially identical,
the higher intensity of the former means that when
merged the green fluorescence was dominant in some
cells.
Protein import into isolated mitochondria
None of the thiolases were apparently targeted to
mitochondria in vivo. However, it is possible that up-
take was prevented by the GFP fusion, or that mito-
chondria normally take up less thiolase than
peroxisomes, such that GFP fluorescence from mito-
chondria did not reach an intensity to be detected. To
test these possibilities we examined the ability of iso-
lated mitochondria to take up all nine thiolases. Each
protein was synthesized in a rabbit reticulocyte lysate
translation system in the presence of radiolabelled
methionine, and then tested for import into mito-
chondria isolated from Arabidopsis cell cultures. As a
control the import and processing of AOX and Rubisco
SSU were examined, the former as a positive control
for import and the latter as a control to demonstrate
the specificity of import into isolated mitochondria
(Chew et al. 2003; Chew and Whelan 2004). In this
case the AOX precursor protein (36 kDa) was
imported and cleaved to a mature protein (32 kDa) as
previously demonstrated (Fig. 3, lanes 1 and 2)
(Whelan et al. 1995). Addition of protease resulted in
the 32-kDa mature form being resistant to protease
digestion indicating uptake by mitochondria. This
resistance was abolished by the addition of valinomy-
cin that dissipates the inner membrane potential
(Fig. 3, lanes 4 and 5) (Tanudji et al. 2001). The
phosphate translocator from maize was used as an
additional control, after uptake into mitochondria and
rupture of the outer membrane protease digestion
results in a small cleaved protected fragment of
33 kDa, indicating that the added protease has access
to inside the outer membrane (Bathgate et al. 1989;
Murcha et al. 2004, 2005; Winning et al. 1992). In
contrast to the mitochondrial controls, Rubisco SSU
was not protected from protease digestion indicating it
was not imported into mitochondria (Fig. 3).
When the nine thiolase proteins were tested for
mitochondrial uptake two distinct patterns were ob-
served, KAT1, ACAT 1.1, ACAT 1.2, ACAT 1.3,
KAT5.1 and KAT5.2 did not yield any protease pro-
tected products upon incubation with mitochondria
and thus were deemed not to be imported (Fig. 3).
KAT2, ACAT2.1 and ACAT2.2 yielded resistant
products upon incubation with mitochondria. Although
KAT2 was not proteolytically cleaved by mitochon-
dria, a protease resistant product with a lower mol
mass was obtained when mitochondria were treated
with Proteinase K (Fig. 3, lanes 1–3). Notably this was
also generated in the presence of valinomycin (Fig. 3,
lanes 4–5). However, upon rupture of the outer
Table 1 Summary of the subcellular location of thiolase proteins
Protein Locus Target prediction Peroxisomaltargeting
Proteomic In vivo In vitro Location Function
KAT 1 At1g04710 M PTS2 P NM PeroxisomeKAT 2 At2g33150 M PTS2 Ma,b, Cc, Nd P NM Peroxisome b-oxidationKAT 5.1 At5g48880.1 M – NT NM Cytosol Flavonoid biosynthesisKAT 5.2 At5g48880.2 M PTS2 P NM Peroxisome Flavonoid biosynthesisACAT 1.1 At5g47720.1 None – NT NM CytosolACAT 1.2 At5g47720.2 None – NT NM CytosolACAT 1.3 At5g47720.3 None – P NM PeroxisomeACAT 2.1 At5g48320.1 None – NT NM Cytosol Mevalonate pathwayACAT 2.2 At5g48230.2 None – NT NM Cytosol Mevalonate pathway
Targeting prediction = M (mitochondria) if two or more predictions indicate a mitochondrial location. Peroxisomal Targeting = thepresence of a Type 1 or 2 peroxisomal targeting signal. Proteomic = evidence for location from independent proteomic studies,M = Mitochondria, C = chloroplast and N = nuclear.a,b Kruft et al (2001) and Heazlewood et al (2004), c Kleffmann et al (2004), d Pendle et al (2005). In vivo = targeting ability as byGFP tagging, P = peroxisomal and NT = no targeting. In vitro tested ability to target to mitochondria, NM = not taken up intoisolated mitochondria. Final column indicates the location concluded and suggested role in metabolism
102 Plant Mol Biol (2007) 63:97–108
123
membrane this product was absent (Fig. 3, lanes 6–9).
In the case of ACAT2.1 and ACAT2.2 a similar pat-
tern was observed except that the protected protein
had the same molecular mass as the protein added to
the import assay (Fig. 3, lanes 1–5). Again with rupture
of the outer membrane no protease protection was
observed (Fig. 3, lanes 6–9).
Although the protease protected fragments pro-
duced upon incubation of KAT2, ACAT2.1 and
ACAT2.2 may suggest uptake by mitochondria, their
presence when valinomycin was added to the import
assay and their sensitivity when the outer mitochon-
drial membrane is ruptured suggests that they may
represent protease resistant products in the presence of
intact mitochondria. The protease susceptibility of
KAT2, ACAT2.1 and ACAT2.2 was tested by the
ability of added protease to digest the radiolabelled
precursor protein. Incubation of KAT2.1, ACAT2.1
and ACAT2.2 with proteinase K alone indicated that
they were resistant to protease digestion; in contrast
AOX was completely digested (Fig. 4). Thus it was
concluded that there was no uptake of any radiola-
belled thiolase proteins into isolated mitochondria, in
agreement with the GFP targeting (Fig. 2).
Co-expression analysis
The probable functions of these type I and type II thio-
lases in their determined subcellular locations was
examined by analysis of co-expression of these genes in
microarray data from Arabidopsis (Fig. 5). We used the
Expression Angler co-expression correlation tool from
the Botany Array Resource (Toufighi et al. 2005) to find
the most co-expressed genes based on microarray
hybridization data on Arabidopsis 22K genechips; the
top 25 co-expressed genes are shown in each case
(Fig. 5A, Supplementary Table 1). This analysis shows
that KAT2 co-expresses (Correlation >0.65–0.79) more
highly with a range of peroxisomal fatty acid degrada-
tion components in the peroxisome than with any other
nuclear genes in Arabidopsis. These included the fatty
acid multifunction protein MFP2 (At3g06860), citrate
synthase (At2g42790), acyl-CoA oxidases (At5g65110,
At3g51840) and enoyl-CoA hydratase (At4g16210)
Fig. 2 In vivo targeting ability of thiolases in Arabidopsis. ThecDNA coding sequences of thiolase from Arabidopsis weretagged with GFP to assess targeting ability. Each cell shown wastransformed with both a GFP construct and with RFP with a typeI PTS. Each panel shows the localization of GFP targeted eitherby the mitochondrial protein alternative oxidase (AOX) or bythiolases (GFP panel). A peroxisomal pattern obtained in thesame cell with the RFP with a type I PTS is shown (RFP-SRLpanel) together with the merged images (Merged panel)
bGFP RFP-SRL Merged
AOX
KAT1At1g04710
KAT2At2g33150
ACAT 1.1At5g47720.1
ACAT 1.2At5g47720.2
ACAT 2.1At5g48230.1
ACAT 2.2At5g48230.2
KAT 5.1At5g48880.1
KAT 5.2At5g48880.2
ACAT 1.3At5g47720.3
20 µm
Plant Mol Biol (2007) 63:97–108 103
123
(Fig. 5A). Curiously, Expression Angler showed KAT5
is not co-expressed with b-oxidation enzymes, but
instead with a series of flavonoid biosynthesis enzymes
(Correlation >0.6–0.71), including flavanone 3-hydrox-
ylase (At3g51240) 4-courmarate CoA ligase
(At1g65060), chalcone synthase (At5g13930), chalcone
isomerase (At5g05270). Notably this pathway requires
short acyl-CoAs for biosynthesis.
The gene encoding a type II enzyme ACAT2
(At5g48230) was found by Expression Angler to be co-
expressed with a range of genes, but notably, hydrox-
ymethylglutaryl-CoA synthase (At4g11820) and
mevalonate diphosphate decarboxylase (At2g38700,
At3g54250) were highly co-expressed (Correlation
>0.80) (Fig. 5, Supplementary Table 1). This is con-
sistent with the role of type II genes in the cytosolic
mevalonate pathway leading to isoprene-containing
compounds such as sterols and terpenoids.
The isoleucine catabolism pathway involves the
branched chain amino acid dehydrogenase complex
(At5g09300, At3g13450, At3g06850), isovaleryl-CoA
dehydrogenase (At3g45300), enoyl-CoA hydratase
(At4g31810) in mitochondria, and then 3-hydroxy-2-
methylbutyryl-CoA dehydrogenases and the fatty acid
multifunction proteins (At4g29010, At3g06860,
At3g15290), in addition to a thiolase, but these fore-
mentioned genes do not appear to be co-expressed
with any of the thiolase genes (data not shown).
pAOX
KAT 2At2g33150
ACAT 2.1At5g48230.1
ACAT 2.2At5g48230.2
36 kDa
48 kDa
40 kDa
41 kDa
PKPrecursor
Lane 1 2+ +
+-
Fig. 4 Protease susceptibility of KAT 2, ACAT 2.1 and ACAT2.2. The ability of proteinase K to digest thiolases was tested byincubating the protease with radiolabelled protein. Alternativeoxidase was used as a control and apparent mol mass areindicated in kDa
ValPK
Mit-OMMit
Lane-
+-
1 2 3 4 5 6 7 8 9
pAOX
pPic
KAT 1 At1g04710
pTIM23
pRubisco SSU
mAOX
mPic
KAT 2At2g33150
ACAT 1.1At5g47720.1
ACAT 1.2At5g47720.2
ACAT 2.1At5g48230.1
ACAT 2.2At5g48230.2
KAT 5.1At5g48880.1
KAT 5.2At5g48880.2
46 kDa
48 kDa
42 kDa
43 kDa
40 kDa
41 kDa
43 kDa
48 kDa
36 kDa
32 kDa
20 kDa
14 kDa
38 kDa34 kDa33 kDa
20 kDa
---
-- -
-- - - - -- -- - -
-
+ + + ++ + +
+ + + ++ + + +
ACAT 1.3At5g47720.3 43 kDa
Fig. 3 In vitro import of radiolabelled thiolase proteins intomitochondria isolated from Arabidopsis. Lane 1, precursorprotein alone. Lane 2, precursor protein incubated withmitochondria under conditions that support import into mito-chondria. Lane 3, as lane 2 with proteinase K added afterincubation of precursor with mitochondria. Lane 4 and 5, as lane2 and 3 with valinomycin added to the import assay prior to theaddition of precursor protein. Lanes 6–9 as 2–5 except that themitochondrial outer membrane was ruptured after the incuba-tion period with precursor protein but prior to addition ofproteinase K. Apparent mol mass are indicated in kDa.Abbreviations: Mit = mitochondria, Mit-OM = mitochondriawith outer membrane ruptured, PK = proteinase K, Val = vali-nomycin, AOX = alternative oxidase, Pic = phosphate carrier,Rubisco SSU = small subunit of ribulose-1, 5 bisphosphatecarboxylase/oxygenase, p = precursor protein band, m = matureprotein band
104 Plant Mol Biol (2007) 63:97–108
123
To confirm the co-expression groups in Fig. 5A, we
used Genevestigator (Zimmermann et al. 2004) to
cluster KAT2, KAT5 and ACAT2 and their cohort of
highlighted co-expressed genes across a series of
microarray data based on tissue specific expression
(Fig. 5B). This bootstrapped cluster tree showed three
separate groupings of genes, confirming the Expression
Angler analysis of distinct expression patterns of these
three thiolases, correlating with distinct roles in
metabolism. Note this type of expression analysis
cannot distinguish differential roles for isoforms of
proteins resulting from alternative splicing as the probe
sets used to determine expression do not distinguish
between splice forms.
Discussion
Table 1 summarizes the results of our knowledge on
the subcellular localization of thiolase protein in
Arabidopsis. Although some thiolase proteins contain
a predicted mitochondrial targeting signal both in vitro
and in vivo protein localization assays indicate that
they are not imported into mitochondria. This conflicts
with proteome analysis of isolated mitochondria sug-
gesting a mitochondrial localization for KAT2
(Heazlewood et al. 2004; Kruft et al. 2001). We pro-
pose that this is due to contamination by peroxisomal
proteins and that KAT2 is not an authentic mito-
chondrial protein. Heazlewood et al (2004) reported a
low level of contamination of their mitochondrial
samples with peroxisomes, consistent with the poten-
tial for some false positive identifications in this shot-
gun proteomic study. The apparent requirement of a
thiolase for isoleucine catabolism in mitochondria
(Taylor et al. 2004) is not a strong argument for the
role of KAT2 in mitochondria, as mitochondrial type I
enzymes in animals are structurally distinct from
Arabidopsis KATs (Fig. 1). The terminal step and
isoleucine degradation might be best served by the
type II rather than a type I enzyme, and we have now
shown convincingly that type II thiolases are in the
cytosol and/or peroxisome in Arabidopsis (Fig. 2).
Our data suggests that at least in Arabidopsis,
thiolases involved in b-oxidation are not present in
mitochondria, despite the fact that some biochemical
evidence has suggested this may take place in mito-
chondria of pea (Masterson and Wood 2001). The
results presented here however cannot be definitive for
all plant species as it is possible that genes encoding
thiolases in other plant species may have mitochondrial
targeting ability due to the fact that at least some
thiolase genes in Arabidopsis encode proteins that
have predicted mitochondrial targeting ability. Thus
relatively small changes are likely required to achieve
mitochondrial targeting of plant thiolases, as has been
observed with some peroxisomal thiolases from other
organisms (Danpure et al. 2003; Tsukamoto et al.
1994).
0.5
0.6
0.7
0.8
0.9
1
At3g0
6860
At2g4
2790
At5g6
5110
At4g1
6210
At3g5
1840
0.5
0.6
0.7
0.8
0.9
1
At3g5
1240
At5g0
5270
At5g1
3930
KAT5 (At5g48880)
0.5
0.6
0.7
0.8
0.9
1
At4g1
1820
At2g3
8700
At3g5
4250
Acyl-CoA oxidase (At3g51840)Acyl-CoA oxidase (At5g65110)KAT2 (At2g33150)Citrate synthase (At2g42790)Enoyl-CoA hydratase (At4g16210)Fatty acid multifunction protein MFP2 (At3g06860)Flavanone 3-hydroxylase (At3g51240)Chalcone synthase (At5g13930)Chalcone isomerase (At5g05270)4-courmarate CoA ligase (At1g65060)KAT5 (At5g48880)Mevalonate diphosphate decarboxylase (At2g38700)ACAT2 (At5g48230)Hydroxymethylglutaryl-CoA synthase (At4g11820)Mevalonate diphosphate decarboxylase (At3g54250)
100%
100%
KAT2 (At2g33150)
At1g6
5060
ACAT2 (At5gt48230)
A
B
70%
100%
Fig. 5 In silico expression analysis. (A) The 25 genes co-expressed to the greatest extent with each thiolase gene weredetermined using the Expression Angler tool from the BotanyArray Resource. The annotated genes indicated for KAT2,KAT5 and ACAT2 are those for which the proteins encoded bythese genes could function in peroxisomal fatty acid degradation,flavonoid biosynthesis and the mevalonate pathway, respectively.(B) Clustering analysis of co-expressed genes with KAT2, KAT5and ACAT2 to determine which (KAT or ACAT) branch withco-expressed genes as determined by Expression Angler. EachKAT or ACAT is located in a distinct group in agreement withanalysis by Expression Angler, with good bootstrap valuessupporting the branch points
Plant Mol Biol (2007) 63:97–108 105
123
The data on enzymes required for isoleucine deg-
radation from 2-methyl-3-hydroxybutyryl-CoA
through to propionyl-CoA increasingly suggests that
this part of the biochemical pathway is a non-mito-
chondrial activity. Of the three 3-hydroxy-2-methyl-
butyryl-CoA dehydrogenases in Arabidopsis
(At4g29010, At3g06860, At3g15290), At3g06860 has
been located to peroxisomes by three separate reports
using GFP tagging (Cutler et al. 2000; Koh et al. 2005;
Tian et al. 2004) and At3g15290 has been located to
chloroplasts by mass spectrometry (Kleffmann et al.
2004). The type II ACAT thiolases are all non-mito-
chondrial (Fig. 2), being present in either the cytosol
or peroxisome from our own data. Transport of
2-methyl-3-hydroxybutyryl-CoA out of mitochondria
has not been investigated, but the substrate specificity
of an array of known mitochondrial carriers from the
Mitochondrial Carrier Protein (MCP) family, the
Preprotein and Amino acid Transporter (PRAT)
family and ATP Binding Cassette (ABC) transporters
remain to be studied in Arabidopsis (Pohlmeyer et al.
1997; Rassow et al. 1999; Brugiere et al. 2004; Picault
et al. 2004). The distribution of pathways of amino
acid biosynthesis and metabolism between organelles
and the cytosol is relatively common in plants, but in
the case of isoleucine metabolism, although the met-
abolic enzymes involved are now relatively clear, the
transport activities that facilitate this pathway be-
tween mitochondria, the cytosol and the peroxisome
remain to be elucidated.
Co-expression analysis of transcript data can be a
powerful tool to confirm other data or provide leads
for further analysis. In this case, the co-expression
results for KAT2 are consistent with all our
experimental data. This gene encodes a peroxisomal
thiolase and is co-expressed with other peroxisomal
proteins involved in the same process, namely
b-oxidation of fatty acids. For ACAT2 the subcellu-
lar location, enzyme class and co-expression also
coincide to suggest a role in mevalonate biosynthesis
leading to isoprenes. The KAT5 co-expression result
was a surprise as this protein was suspected to be
involved in b-oxidation based on its enzyme class.
However, the different location of KAT5.1 and
KAT5.2 (Table 1), the fact that KAT5 does not
maintain b-oxidation in seedlings of the KAT
knockout but can partially complement for the lack
of KAT2 when driven by 35S expression (Germain
et al 2001), and the co-expression link with flavonoid
biosynthesis rather than b-oxidation genes (Fig. 5),
suggests that while KAT5 encodes a thiolase, it has a
distinct role to KAT2 in acyl-CoA metabolism in
plants.
Acknowledgements This work was funded through grants fromthe Australian Research Council (ARC) Centre of ExcellenceProgramme to JW, SS and AHM. AHM is funded as an ARCQueen Elizabeth II Fellow and SS as an ARC Federation Fellow.
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Chapter 3 Approaches to defining dual targeted proteins
40
Chapter 3
Approaches to defining dual targeted proteins
Chapter 3 Approaches to defining dual targeted proteins
41
Foreword to Study II Study I revealed that dual targeted proteins can be difficult to determine even
with substantial experimental evidence. To date, most dual targeted proteins identified
have been identified in single or small gene family studies. With the exception of
aminoacyl-tRNA synthetases, there has been no genome wide, systematic study to
identify dual targeted proteins in Arabidopsis thaliana (Duchene et al., 2005). Study II
aimed to take a broader view of dual targeting in Arabidopsis by seeking to identify a
large number of dual targeted proteins. A list of candidates for dual targeted proteins
was generated using computational prediction of subcellular location of proteins (Small
et al., 2004), cross-over sets in subcellular proteomes of mitochondria, chloroplasts and
peroxisomes (Heazlewood et al., 2007) and a list of proteins predicted to be located in
the mitochondria and the nucleus (Schwacke et al., 2007). The subcellular localisation
of a number of these candidate proteins were then tested experimentally.
Using this approach a total of 12 new dual targeted proteins were identified in
Arabidopsis. Five proteins were dual targeted to mitochondria and plastids, six were
dual targeted to mitochondria and peroxisomes, and one that was dual targeted to
mitochondria and the nucleus. In the course of this analysis, it became evident that a
number of technical parameters need to be taken into account when performing
subcellular localisation experiments using GFP fusions. First, the position of GFP with
respect to the tagged polypeptide is important, (e.g. N or C-terminal tagging is
important, as only analysing one over the other, can mask organelle targeting signals).
Second, the segment of the candidate protein chosen to be fused with GFP can also
greatly affect the results obtained. Third, the use of different tissue types or cells was
found to affect the level of dual targeting observed. It should also be noted that testing
of all available gene models is also required if more than one model exists for a gene
locus.
TECHNICAL ADVANCE
Approaches to defining dual-targeted proteins in Arabidopsis
Chris Carrie, Kristina Kuhn, Monika W. Murcha, Owen Duncan, Ian D. Small, Nicholas O’Toole and James Whelan*
ARC Centre of Excellence in Plant Energy Biology, University of Western Australia, MCS Building M316, 35 Stirling
Highway, Crawley 6009, WA, Australia
Received 31 July 2008; revised 25 October 2008; accepted 30 October 2008; published online 12 December 2008.*For correspondence (fax +61 8 64884401; e-mail [email protected]).
Summary
A variety of approaches were used to predict dual-targeted proteins in Arabidopsis thaliana. These predictions
were experimentally tested using GFP fusions. Twelve new dual-targeted proteins were identified: five that
were dual-targeted to mitochondria and plastids, six that were dual-targeted to mitochondria and
peroxisomes, and one that was dual-targeted to mitochondria and the nucleus. Two methods to predict
dual-targeted proteins had a high success rate: (1) combining the AraPerox database with a variety of
subcellular prediction programs to identify mitochondrial- and peroxisomal-targeted proteins, and (2) using a
variety of prediction programs on a biochemical pathway or process known to contain at least one dual-
targeted protein. Several technical parameters need to be taken into account before assigning subcellular
localization using GFP fusion proteins. The position of GFP with respect to the tagged polypeptide, the tissue
or cells used to detect subcellular localization, and the portion of a candidate protein fused to GFP are all
relevant to the expression and targeting of a fusion protein. Testing all gene models for a chromosomal locus is
required if more than one model exists.
Keywords: Arabidopsis, dual targeting, mitochondria, chloroplasts, peroxisomes, nucleus.
Introduction
An essential step in defining the function of any protein is
clarifying its subcellular location. The completely sequenced
genomes of plants such as Arabidopsis thaliana (Arabidop-
sis Genome Initiative, 2000) (2005), Physcomitrella patens
(Rensing et al., 2008) and Chlamydomonas reinhardtii
(Merchant et al., 2007) and extensive ESTs from various
plant species encode many proteins for which the function
is either unknown or is annotated based on sequence
similarity alone. However, the function of any protein in
eukaryotic cells is not solely a definition of its catalytic
activity; rather it is the product of the catalytic activity and
the subcellular location of the protein. Within a plant cell,
mitochondria and chloroplasts have many enzymatic steps
in common. Both contain ATP synthase complexes with
similar subunits, a partly shared genetic apparatus, and a
variety of related enzymes involved in metabolism and
REDOX biology (Buchanan et al., 2002). Even though many
proteins in these two organelles may have identical catalytic
activity, their cellular function depends on their location.
Various approaches have been used to define the location
of proteins, from computational prediction (Chou and Shen,
2007), GFP tagging (Heazlewood et al., 2007; Koroleva et al.,
2005) and subcellular proteomics (Heazlewood et al., 2007;
Lilley and Dupree, 2007) to direct studies on individual
gene products. One disadvantage evident with large-scale
approaches is that they usually are ‘winner takes all’
approaches, and often define a single location for any given
protein. Each approach has limitations, e.g. computational
prediction usually only works reliably for soluble proteins
with clear organelle-targeting signals, GFP tagging may
result in artefacts, such as accumulation in the nucleus, or
alterations in the targeting ability of the protein due to a
foreign passenger protein (Chew et al., 2003a), while sub-
cellular proteomics often focuses on one organelle, and
proteins ‘claimed’ by two organelles are left unresolved
(Heazlewood et al., 2007).
Defining location is even more complicated for pro-
teins that are located in two compartments, so-called
1128 ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd
The Plant Journal (2009) 57, 1128–1139 doi: 10.1111/j.1365-313X.2008.03745.x
dual-targeted proteins. The first dual-targeted protein
reported in plants was glutathione reductase from Pisum
sativum (pea) (Creissen et al., 1995). Since then, the list of
dual-targeted proteins has grown to approximately 40 from
a variety of species (Duchene et al., 2005; Mackenzie, 2005;
Peeters and Small, 2001; Silva-Filho, 2003). Dual targeting of
a protein can be achieved in a number of ways (Peeters and
Small, 2001; Regev-Rudzki and Pines, 2007; Silva-Filho,
2003): (1) via alternative transcriptional initiation to provide
a protein with different targeting signals, e.g. glutathione
S-transferase F8 (Thatcher et al., 2007), (2) via a targeting
signal directing a protein to two locations, referred to as an
ambiguous targeting signal, e.g. glutathione reductase
(Chew et al., 2003a; Creissen et al., 1995), (3) by two different
targeting signals within one polypeptide, e.g. catalase A in
yeast (Petrova et al., 2004), (4) through utilization of alter-
native translation start sites, currently suggested for a DNA
polymerase targeted to mitochondria and chloroplasts
(Christensen et al., 2005), and also for holocarboxylase
synthetase 1 located in mitochondria and the cytosol
(Puyaubert et al., 2007), and (5) via retrograde translocation,
best known in yeast in which a single fumarase gene
encodes both the mitochondrial and cytosolic forms of
fumarase (Regev-Rudzki and Pines, 2007).
Defining the subcellular localization of a protein with
accuracy is important, as large-scale phenotyping screens,
microarray experiments and protein–protein interaction
assays all rely on such information to build hypotheses or
define models. However, in the case of dual-targeted
proteins, there was no systematic analysis or approach to
define such proteins. With the exception of aminoacyl-tRNA
synthetases (Duchene et al., 2005), most dual-targeted pro-
teins identified to date were identified in studies on individ-
ual proteins. One potential drawback of such approaches is
that dual-targeted proteins may be missed or ascribed to a
single organelle.
Here we describe an approach to define dual-targeted
proteins in Arabidopsis thaliana. Lists of proteins that are
candidates for dual targeting have been generated using
computational prediction of the subcellular location of
proteins (Small et al., 2004), from cross-over sets in subcel-
lular proteomes of mitochondria, chloroplast and peroxi-
somes (Heazlewood et al., 2007), and from a set of proteins
predicted to be located in mitochondria and the nucleus
(Schwacke et al., 2007). We experimentally tested the sub-
cellular localization of subsets of these proteins, thereby
defining dual-targeted proteins found in mitochondria and
peroxisomes, mitochondria and plastids, and mitochondria
and the nucleus. We observed that the tissue type used to
test for dual targeting can greatly affect results, and that both
N- and C-terminal GFP tagging of the investigated polypep-
tide as well as co-transformation of GFP constructs with
appropriate organelle-targeted RFP control constructs are
essential to accurately define subcellular location.
Results
Various approaches were used to computationally predict
dual-targeted proteins in Arabidopsis. To predict dual tar-
geting to mitochondria and plastids, we used the neural-
network-based program Predotar (Small et al., 2004). The
Predotar networks were not trained using any dual-targeted
proteins, and thus the mitochondrial and plastid scores from
the networks tend to be antagonistic. Indeed, the majority of
known dual-targeted plastid/mitochondrial plant proteins
are predicted as uniquely plastidial by Predotar. Therefore,
the complete set of annotated Arabidopsis protein
sequences were run through Predotar twice, once using the
plant prediction networks and once using animal/fungal
prediction networks that were not trained with plastid
sequences. A total of 803 protein sequences were predicted
to contain a plastid-targeting sequence by the plant networks
and to contain a mitochondrial-targeting sequence by the
animal/fungal networks (Table S1a). This list contains 15
proteins that were previously reported to be dual-targeted in
Arabidopsis, which represents a significant enrichment
compared to the number of known dual-targeted proteins
expected to occur in a random group of this size of organelle-
targeted proteins (Table 1), suggesting that this approach is
useful in narrowing down candidates. The dual-targeting
candidates were then ranked by their mitochondrial score
using the animal/fungal networks minus the mitochondrial
score using the plant networks. This ranking places most of
the known dual-targeted proteins towards the top of the list
(Table S1a). As a second strategy, we used available
proteome datasets to predict dual targeting. The reported
proteomes of mitochondria and chloroplasts have an over-
lap of 97 proteins (Arabidopsis subcellular proteomic (SUBA)
database; Heazlewood et al., 2007 and references therein),
three of which have been previously reported to be dual-
targeted (Table S1b), again significantly more than expected
in any random group of 100 proteins (Table 1). In a third
computational approach, all known and putative peroxi-
somal proteins in the Araperox database (Reumann et al.,
2004) were cross-referenced against proteins that had been
experimentally identified in mitochondria or chloroplasts or
that were predicted to be mitochondrial or plastidial using 10
prediction programs. This produced a list of nine proteins
(Table S1c). As there are no known proteins targeted to
mitochondria and peroxisomes, it was not possible in
advance to determine the significance of these predictions.
To further identify potentially dual-targeted proteins, we
focused on processes that occur in different organelles,
specifically DNA replication and transcription. Several
known dual-targeted proteins, such as two DNA polymer-
ases (Christensen et al., 2005; Elo et al., 2003), a RecA
homologue (Shedge et al., 2007) and an RNA polymerase
(Hedtke et al., 2000), which are targeted to both mitochon-
dria and chloroplasts, fit into this functional group. From
Dual targeting of proteins in Arabidopsis thaliana 1129
ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 1128–1139
computational screens of the Arabidopsis genome using
publicly available information resources and databases (see
Experimental procedures), we identified seven additional
uncharacterized genes encoding proteins potentially
involved in DNA replication, maintenance or structural
organization that were predicted to be mitochondrial or
plastidial according to the Predotar and TARGETP programs
(Emanuelsson et al., 2000; Small et al., 2004). This group of
proteins was selected for experimental studies. Eleven
transcription factors that have been predicted, but as yet not
experimentally confirmed, to be located in mitochondria as
well as in the nucleus were also tested (Schwacke et al.,
2007) (Table S1d,f).
Taken together, all computational approaches produced a
non-redundant list of 905 proteins, 19 of which have
previously been reported to be dual-targeted. In order to
experimentally test the predicted dual targeting, we gener-
ated GFP reporter constructs for 41 of these proteins. The
Predator list of 803 was too long to test systematically, so we
chose five proteins from the top 20 and 10 more throughout
the list, allowing assessment of the rank order (Supplemen-
tary Table 1A). For the ‘Mass spec’ list of 97 proteins that
have been determined to be located in mitochondria and
plastids (Table 1, Supplementary Table 1B), we tested 10 of
these proteins, and for the other lists we tested more than
50% of the proteins. In the case of the Predator and ‘Mass
spec’ lists, the proteins were chosen at random within the
parameters outlined above. Mitochondrial and plastid
targeting were tested using C-terminal tagging with GFP,
and peroxisomal targeting was studied using N-terminal
tagging. GFP was fused to the full or partial sequence of
candidate proteins, and fluorescence was monitored follow-
ing biolistic transformation of Arabidopsis cell cultures with
these constructs. This approach allowed a relatively large
number of samples to be tested, as each test only requires
2 ml of culture cells on a filter support on an MS agar plate.
When investigating dual targeting to mitochondria and
plastids, Arabidopsis seedlings and onion epidermal cells
were transformed in addition to cell cultures. To define the
subcellular localization of GFP fluorescence with certainty,
we generated RFP control constructs driving expression of
mitochondrial-, plastid- or peroxisomal-targeted RFP (Carrie
et al., 2007; Murcha et al., 2007). For each protein to be
tested, GFP constructs were co-transformed with the appro-
priate RFP control constructs. This avoided using stains such
as Mitotracker to define organelles and provided a positive
control for the success of transformation and protein uptake
into the appropriate organelle. The subcellular localizations
of all proteins tested are shown in Figures 1, 2, 3 and 5 and
Figures S1 and S2.
To experimentally define proteins that were targeted
to mitochondria and peroxisomes, N- and C-terminal GFP
fusions were constructed and targeting was studied for
three proteins (Table S1c). When GFP was placed at the
C-terminus of a carrier protein (At3g55640), malonyl CoA
decarboxylase (At4g04320) or alanine/glyoxylate amino-
transferase (At4g39660), a fluorescence pattern that was
identical to that obtained with mitochondrial RFP was
observed (Figure 1, images 1a–1c, 3a–3c and 5a–5c). How-
ever, when GFP was placed at the N-terminus, a pattern that
overlapped with peroxisomal RFP was obtained (data not
shown). To confirm that the peroxisomal targeting was due
to the predicted peroxisomal-targeting signal type 1 (PTS1)
at the C-terminus of the proteins, the last 10 amino acids of
each of the three proteins were fused to the C-terminus of
GFP (Figure 1, images 2d–2f, 4d–4f and 6d–f). Peroxisomal
GFP fluorescence was again observed. Thus dual targeting
was detected for the three proteins tested from the group of
Table 1 Summary of predicted and exper-imentally determined dual-targeted pro-teins in Arabidopsis
Method ofprediction
Total no.proteins
No. knowndual-targetedproteins inthis list P-values
No.proteinstested
No. knowndual-targetedproteins afterthis study
Predotar 803 15 0.0003 14 17Mass spec 97 3 0.08 11 4AraPerox 9 0 N/A 6a 6a
Transcription factors 11 0 N/A 8 1DNA maintenance 9 2 N/A 9 5Totals 905 19 N/A 41a 31a
A number of approaches were used to predict dual-targeted proteins in Arabidopsis. The totalnumber of proteins predicted by each approach to be dual-targeted and the number ofexperimentally verified dual-targeted proteins is indicated for each approach. The enrichment ofdual-targeting proteins in each list was determined compared to groups of randomly selectedmitochondrial and chloroplasts proteins as outlined in Experimental procedures. The targetingability of the number of proteins tested from each group and the number of dual-targetedproteins from each group after this study are also shown. The totals represent the non-redundantnumbers tested due to overlap between groups.aThis group includes three alternative NAD(P)H dehydrogenases that we have shown to bedual-targeted to mitochondria and peroxisomes (Carrie et al., 2008).
1130 Chris Carrie et al.
ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 1128–1139
putative peroxisomal and mitochondrial proteins, but this
depended on the nature of the GFP construct.
When testing the targeting properties of the seven
proteins that may function in DNA maintenance, replication
or transcription, we detected dual targeting to mitochondria
and chloroplasts for three proteins, a topoisomerase I TopI-
like protein (At4g31210, AtTopI), a protein with similarity to
the human mitochondrial DNA helicase Twinkle (At1g30680,
AtTwinkle) and a protein related to human RCC1 and
Arabidopsis Uvr8 (At5g08710; RFP tagging was used for
this gene for cloning reasons) (Figure 2, image series 3, 4
and 5). For AtTopI, as for the dual-targeted DNA polymerase
Polc1 (Elo et al., 2003), which we used as a control (Fig-
ure S2, image series 1), the intensity of fluorescence from
mitochondria and plastids was equal in most transformed
suspension cells. For AtTwinkle, the plastid fluorescence
was much stronger than mitochondrial fluorescence in
suspension cells and onion cells, but the intensity of
fluorescence from mitochondria was equal to that from
plastids in Arabidopsis seedlings (Figure 2, images series 3).
The different intensities of GFP fluorescence in the two
organelles were not restricted to this group of proteins.
When we tested the targeting of calcium-sensing receptor
(CaS, At5g23060), a phosphoprotein that has been shown to
be located in thylakoid membranes (Vainonen et al., 2008)
but is potentially dual-targeted according to Predotar and
organelle proteome overlap (Table S1), dual targeting was
observed with onion epidermal cells and with Arabidopsis
seedlings, but not with suspension cell cultures (Figure 2,
image series 6). We previously observed a similar tissue-
specific variation of GFP fluorescence for dual-targeted
type II NAD(P)H dehydrogenases (Figure S2, image series
2) (Carrie et al., 2008). Thus the ability to detect dual
targeting depended on the nature of the tissue.
The above findings prompted us to analyse the targeting
ability of DNA polymerase c2 (Polc2), which has been
MergedRFPSRLMergedAOXRFP
10 µm
GFP AA 1-518
GFP AA 322-332
GFPAA 1-332
GFP AA 466-476
GFP AA 1-476
GFP AA 508-518
Alanine/Glyoxylate aminotransferase (AGAT)
(At4g39660)
Malonyl CoA decarboxylase (MDC)(At4g04320)
Carrier protein (CP)(At3g55640)
Test for mitochondrial targeting ability Test for peroxisomal targeting ability
3a 3b 3c 3d 3e 3f
4a 4b 4c 4d 4e 4f
5a 5b 5c 5d 5e 5f
6a 6b 6c 6d 6e 6f
1a 1b 1c 1d 1e 1f
2a 2b 2c 2d 2e 2f
Figure 1. Proteins found to be dual-targeted to mitochondria and peroxisomes.
N- and C-terminal GFP fusion proteins were constructed for proteins predicted to be located in mitochondria and peroxisomes. The amino acids (AA) fused to GFP
are indicated in a schematic representation of each construct (left). Targeting ability was tested in Arabidopsis suspension cells using AOX–RFP as a control for
mitochondrial targeting and RFP–SRL as a peroxisome-specific control.
Dual targeting of proteins in Arabidopsis thaliana 1131
ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 1128–1139
previously reported as a dual-targeted protein (Christensen
et al., 2005). Christensen et al. (2005) detected targeting
exclusively to plastids if translation was initiated at the first
in-frame AUG codon. When using a construct that included
the annotated 5¢ UTR, dual targeting was observed, which
was proposed to be due to translation starting at a CUG
codon seven amino acids upstream of the AUG (Christensen
et al., 2005). The authors proposed that the targeting of
Polc2 is regulated by alternative translation initiation. In
contrast, we observed that, when translation started at the
first in-frame AUG, GFP was targeted to both mitochondria
and plastids, even though mitochondrial fluorescence was
much weaker than plastid fluorescence in suspension cells
(Figure 3a, image series 1 and 2). Mitochondrial GFP fluo-
rescence was detected in onion cells and Arabidopsis
seedlings (Figure 3b, image series 4), and exclusive mito-
chondrial localization was occasionally observed in Arabid-
opsis seedlings (Figure 3b, image 4c). Addition of seven
amino acids was performed as described by Christensen
et al. (2005), resulting in targeting to both organelles with
equal intensity of fluorescence in Arabidopsis cell suspen-
sions and onion cells (Figure 3a,b), but only to mitochondria
in Arabidopsis seedlings (Figure 3b, image 5b).
To further study the import of Polc2 into mitochondria
and plastids, we performed in vitro import assays on the
DNA polymerase protein starting with the standard AUG
codon. Although such large proteins are usually difficult
to study with in vitro import assays, we readily detected
the import of this protein into mitochondria isolated from
Arabidopsis seedlings (Figure 4a, left panel). Import
was abolished by addition of valinomycin to the import
assay, indicating that it was dependent on the membrane
potential, as expected for a protein translocated into the
mitochondrial matrix. No import of the precursor of the
small subunit of 1,5-ribulose bisphosphate carboxylase/
oxygenase (Rubisco SSU) into isolated mitochondria was
observed (Figure 4b, right panel), indicating that the
import of Polc2 observed was not due to plastid contam-
ination or non-specific import of chloroplast proteins into
isolated mitochondria, as previously reported for isolated
pea mitochondria (Cleary et al., 2002), but not for isolated
Arabidopsis mitochondria as used in this study (Chew
et al., 2003b).
We next analysed the kinetics of import into mitochon-
dria isolated from suspension cell cultures and Arabidop-
sis seedlings used in the above import assays (Figure 3b).
Arabidopsis cell culture
MergedSSURFPMergedAOXRFP
ArabidopsisseedlingsOnion cells
RFPAA 1-117
RCC1/Urv8 like protein (434 AA)At5g08710
GFPAA 1-387
CaS like protein (387 AA)At5g23060
GFPAA 1-192
Topoisomerase I (AtTopI) (1280 AA)At4g31210
GFPAA 1-42
Alternative oxidase (AOX) (321 AA)
GFPAA 1-180
Small subunit of 1,5 ribulose bisphosphatecarboylase/oxygenase (SSU Rubisco) (180 AA)
GFPAA 1-184
DNA helicase twinkle (Atwinkle) (709 AA)At1g30680
Test for mitochondrial targeting ability Test for chloroplastidic targeting ability
10 µm
1a 1b 1c 1d 1e 1f 1g 1h
2a 2b 2c 2d 2e 2f 2g 2h
3a 3b 3c 3d 3e 3f 3g 3h
4a 4b 4c 4d 4e 4f 4g 4h
f5e5d5c5b5a5
h6f6e6d6c6b6a6 6g
5g 5h
Figure 2. Proteins found to be dual-targeted to mitochondria and chloroplasts.
Subcellular localization was predicted for a list of proteins involved in DNA maintenance replication and transcription (Table S1d). One other protein (CaS) predicted
to be targeted to mitochondria and plastids was also tested. For the RCC1/Uvr8-like protein, RFP was used as the reporter and GFP as the control; all other candidate
proteins were fused to GFP. The numbers refer to the amino acids used in each construct. The total number of amino acids in each protein is shown after the name if
the full-length protein was not used to make the fusion protein. Targeting ability was tested in Arabidopsis suspension cell cultures, onion epidermal cells and
2-week-old Arabidopsis seedlings.
1132 Chris Carrie et al.
ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 1128–1139
After 10 min, when the import rate was still relatively
linear, the import of alternative oxidase (AOX) was almost
twofold higher into mitochondria isolated from seedlings
compared to cultured cells, and a large difference per-
sisted even after 20 min. For import of Polc2, a difference
of 40% at 20 min was also observed between mitochondria
isolated from seedlings compared to cultured cells. As
equal amounts of mitochondrial protein were used in the
above import assays, we analysed the abundance of
several components of the mitochondrial import appa-
ratus; a representative immunoblot from several mito-
chondrial preparations is shown (Figure 4c). Differences
were observed for some components, e.g. TOM20-2 was
approximately twice as abundant in mitochondria isolated
from cell cultures compared to mitochondria isolated from
plant material. The opposite was seen with TOM20-4. Thus
isolated mitochondria from different tissues import pro-
teins at different rates, which might be related to differ-
ences in the abundance of particular components of the
mitochondrial import apparatus.
A number of transcription factors have been predicted to
be targeted to mitochondria and nuclei in Arabidopsis and
rice (Schwacke et al., 2007). We tested the targeting of
eight of these transcription factors to mitochondria. For
three of these, we initially fused full-length cDNAs to the
GFP coding sequence. With one exception, this resulted
in no GFP fluorescence. A mitochondrial pattern was
obtained for the transcription factor APL (altered phloem
development, At1g79430), indicating that the protein had
mitochondrial-targeting ability (Figure 5a). As GFP fused to
the N-terminus of this protein had been shown previously
to target to nuclei (Bonke et al., 2003), we concluded that
this transcription factor is dual-targeted. For the seven
other transcription factors predicted to possess N-terminal
mitochondrial transit peptides, we fused GFP to the first 60
amino acids of these proteins. GFP fluorescence was seen
Arabidopsis cell culture
GFPAA -7-120
GFPAA 1-120
DNA polymerase γ2 (Polγ2) (1050 AA)At1g50840
DualMitochondrialPlastidDualArabidopsis seedlingsOnion cells
MergedSSURFPMergedAOXRFP
Test for mitochondrial targeting ability Test for chloroplastidic targeting ability
AUG
CUGAUG
1 120
1 120–710 µm
1a 1b 1c 1d 1e 1f
2a 2b 2c 2d 2e 2f
3a 3b 3c 3d 3e 3f
GFPAA -7-120
GFPAA 1-120
DNA polymerase γ2 (Polγ2) (1050 AA)At1g50840AUG
AUG1 120
1 120–7
4a
4b
4c 4d 4e
5a 5b
4b
CUG
(b)
(a)
Figure 3. Targeting of DNA polymerase c2 to mitochondria and chloroplasts.
The targeting ability of the 120 N-terminal amino acids (with the AUG start codon) or the 127 N-terminal amino acids (with the upstream CUG start changed to AUG
to ensure fidelity of translation) of DNA polymerase c2 was tested in three tissue types:
(a) Arabidopsis suspension cell culture and (b) 2-week-old Arabidopsis seedlings and onion epidermal cells. Dual targeting of the native AUG constructs was easier
to visualize in onion cells (image 4a) and Arabidopsis seedlings (images 4d and 4e). In some cases, an exclusive mitochondrial localization was observed in the latter
(image 4c). Dual targeting of the CUG constructs (changed to AUG to ensure fidelity of translation) was observed in Arabidopsis cell suspension and onion cells, but
mitochondrial targeting only was detected in Arabidopsis seedlings (image 5b). The gene models show the sequences used to generate the proteins: the upstream
CUG was converted to AUG, and the native AUG was changed to CUG so that a single protein would be produced in each case. The constructs with the native AUG
did not contain any upstream nucleotides to ensure that translation could only commence at this AUG.
Dual targeting of proteins in Arabidopsis thaliana 1133
ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 1128–1139
mainly in the nucleus and also in the cytosol but never in
mitochondria for these constructs (Figure S1). For two of
the proteins, AtTLP9 (At3g06380) and AtTLP7 (At1g53320),
we cloned full-length cDNAs in order to perform in vitro
import assays into isolated Arabidopsis mitochondria.
No import was detected (data not shown), indicating that
TOM20-2
TOM20-3
TOM20-4
OM64
TOM40
TIM17-2
Plants
Cell cu
lture
Plants Cell culture
Time (min) 2 5 10 20 2 5 10 20
pAOX 36 kDa
mAOX 32 kDa
Lane
MitPKVal
1 2 3 4 5
+ + + ++ +
+ +
–– – –– – –
pAOX 36 kDa
pPolγ2 120 kDa
mAOX 32 kDa
mPolγ2 116 kDa
Lane 1 2 3 4 5MitChlProt + +– – –
+ ++ +–
–– –
– –
pSSU 20 kDa
mSSU14 kDa
pPolγ2 120 kDa
mPolγ2 116 kDa
0.00
0.20
0.40
0.60
0.80
1.00
0 5 10 15 20Time (min)
Plants
AOX
n = 3
0.00
0.20
0.40
0.60
0.80
1.00
0 5 10 15 20
Plants
n = 3
Time (min)
Cell culture Cell culture
Polγ2
(a) (c)
(b)
Figure 4. In vitro import of DNA polymerase c2 into mitochondria, and analysis of the kinetics of protein import into mitochondria from Arabidopsis suspension
cells and seedlings.
(a) In vitro import of DNA polymerase c2 (translated from AUG) into mitochondria. Incubation of the full-length precursor protein with an apparent molecular mass of
120 kDa (lane 1) with mitochondria (lane 2) resulted in a protease-protected band with an apparent molecular mass of 116 kDa (lane 3). The protease protection was
abolished when valinomycin was added to mitochondria prior to the import assay (lanes 4 and 5). A similar pattern was observed with the mitochondrial protein
alternative oxidase. The third panel shows the specificity of import into mitochondria: the precursor of the Rubisco SSU (lane 1) is not imported into a protease-
protected location when incubated with mitochondria (lanes 2 and 3), but is readily imported and protease-protected when incubated with chloroplasts (lanes 4 and 5).
(b) Analysis of the kinetics of protein import into mitochondria isolated from Arabidopsis suspension cell cultures and 2-week-old Arabidopsis seedlings. The
alternative oxidase and Polc2 precursors were incubated with 250 lg of isolated mitochondria from both tissues, and the amount of imported protein was assessed at
various times. The maximum amount of import was set to 1, and other values are expressed as relative amounts. Error bars show standard errors from three
independent uptake experiments.
(c) Western blot analysis of various components involved in the import of proteins into mitochondria: 30 lg of isolated mitochondria were loaded onto each lane.
1134 Chris Carrie et al.
ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 1128–1139
none of the seven transcription factors is targeted to
mitochondria.
Discussion
A number of approaches were used to search for dual-
targeted proteins, leading to identification of 12 new
examples, including proteins targeted to mitochondria and
peroxisomes or mitochondria and nuclei (Table 2). From
these studies, it has emerged that caution must be exercised
when assigning a location to a protein from GFP assays, as a
single targeting location may be deduced when in fact the
protein is dual-targeted or has dual-targeting ability.
The location of the GFP passenger is critical in determin-
ing dual targeting. This was evident for proteins targeted to
mitochondria and peroxisomes via two different signals, an
N-terminal mitochondrial transit peptide and a C-terminal
PTS-1 signal. In mammalian cells, serine:pyruvate/ala-
nine:glyoxylate aminotransferase and 2-methylacyl CoA
racemase have also been shown to target to both mitochon-
dria and peroxisomes via two separate targeting signals, an
N-terminal mitochondrial-targeting signal and a C-terminal
peroxisomal-targeting signal (Amery et al., 2000; Oda et al.,
2000). Alternative transcript initiation has been shown to
produce two mRNAs yielding two different proteins for
serine:pyruvate/alanine:glyoxylate aminotransferase, one of
which lacks the N-terminal mitochondrial-targeting signal
(Oda et al., 2000). A similar mechanism for producing
mitochondrial and peroxisomal proteins from a single gene
may operate in plants. However, 5¢ RACE did not reveal
alternative transcripts for two alternative NAD(P)H dehydro-
genases recently shown to be targeted to mitochondria
and peroxisomes via an N-terminal mitochondrial-targeting
signal and a C-terminal PTS-1 (Carrie et al., 2008).
In the case of the APL transcription factor, two gene
models exist; the longer mRNA has an additional N-terminal
sequence and is not predicted to be targeted to mitochon-
dria, whereas the shorter transcript yields a smaller protein
that is predicted to possess a mitochondrial transit peptide
(Figure 5b,c). Previously, it has been demonstrated that GFP
GFPAA 1-358
APL like transcription factor (358 AA)At1g79430
At1g79430.1
At1g79430.2
Predicted mitochondrialtargeting signal
Nuclear localisationsignal
Locus TargetP MitoProt2 Subloc Ipsort Predotar Mitopred Peroxp Wolfpsort Multiloc LoctreeAt1g79430.1 mito mito nuclear mito mito nuclear mito nuclearAt1g79430.2 nuclear nuclear nuclear nuclear
MergedAOXRFP
10 µm
(a)
(b)
(c)
Figure 5. Analysis of the targeting ability of the transcription factor APL, predicted to be targeted to mitochondria and the nucleus.
(a) When GFP is fused to the C-terminus of the shorter of two proteins encoded by APL (b), only mitochondrial targeting is evident.
(b) Models of two mRNAs that have been proposed for this gene are shown; the positions of a predicted mitochondrial transit peptide and a nuclear localization
signal are shown.
(c) Subcellular targeting for the two forms of APL as predicted by 10 programs.
Table 2 Locations of the dual-tragted proteins in Arabidopsis
Mitochondria/chloroplast Mitochondria/peroxisome Mitochondria/nucleus Chloroplast/nucleus
Dual-targeted proteinsin Arabidopsis
42 (37) 6a (0) 1 (0) 0 (0) 49
The table shows the total number of known dual-targeted proteins in Arabidopsis after this study, and with the number known before this study inparentheses.aThis group includes three alternative NAD(P)H dehydrogenases that we have shown to be dual-targeted to mitochondria and peroxisomes (Carrieet al., 2008).
Dual targeting of proteins in Arabidopsis thaliana 1135
ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 1128–1139
fused to the N-terminus of the larger protein is targeted to
the nucleus (Bonke et al., 2003). We detected mitochondrial
targeting but no nuclear signal when GFP was fused to the
C-terminus of the smaller protein. APL has a predicted
nuclear localization signal (KKRP) starting at amino acid 190
that appears to direct nuclear import only when the
mitochondrial-targeting signal is blocked with additional
amino acids at the N-terminus. Several studies have dem-
onstrated that either removing some N-terminal amino acids
from mitochondrial-targeting signals or placing other amino
acids in front can affect mitochondrial targeting (de Castro
Silva Filho et al., 1996; Chaumont et al., 1994; Rudhe et al.,
2002). For the smaller form of APL in which the mitochon-
drial transit peptide is unmasked, mitochondrial import
appears to be dominant over nuclear targeting. Alternative
splicing may represent a more widely used mechanism to
achieve dual targeting. It has been shown for glutathione
S-transferase F8 that alternative transcript initiation leads to
different subcellular localizations (Thatcher et al., 2007).
Furthermore, improved annotation of genomes often results
in several gene models for a single chromosomal locus.
Thus, although some studies may have determined a single
localization for a protein, newer gene models produced after
these studies must be checked with respect to localization,
as observed here with the APL transcription factor.
We observed that, in the case of dual targeting to
mitochondria and chloroplasts, the tissue used to test
targeting can play a major role in determining whether a
protein is dual-targeted. While some proteins such as
AtTopI and Polc1 were clearly dual-targeted in all three
tissue types tested, dual targeting was difficult to detect for
AtTwinkle, RCC1/Uvr8-like and CaS in Arabidopsis suspen-
sion cells as the fluorescence from mitochondria was
much weaker, but still detectable, than fluorescence from
plastids. However, dual targeting of these proteins was
more readily detected in onion cells and Arabidopsis
seedlings. This finding prompted us to test the targeting
of Arabidopsis Polc2. This protein has previously been
proposed to be dual-targeted by the use of two different
translation start sites, a standard AUG codon and an
upstream CUG (Christensen et al., 2005). We detected dual
targeting with translation starting from the standard AUG
codon in Arabidopsis seedlings, cell culture and onion
cells, and moreover confirmed by in vitro uptake assays
using the full-length Polc2 polypeptide that the protein
translated from the AUG is imported into mitochondria.
These data are consistent with data from tobacco, in which
two DNA polymerases homologous to Polc1 and Polc2
have been reported to be dual-targeted to mitochondria
and plastids when translated from the standard AUG
codon (Ono et al., 2007).
Because we observed differences between different
tissues used to detect dual targeting, we tested the kinetics
of protein import into mitochondria isolated from two
tissue types (Arabidopsis suspension cell cultures and
2-week-old seedlings) in order to determine whether the
import capacity of mitochondria from some tissues affects
the ability to detect dual targeting. We observed that
mitochondria from Arabidopsis seedlings had a faster rate
of import. This may affect the ability to detect dual targeting
as the threshold for GFP fluorescence detection may not be
reached if the rate of mitochondrial protein import is low.
Additionally, an immunoblot analysis of selected compo-
nents of the mitochondrial protein import apparatus
showed that some of the receptor components differed in
abundance between tissues, i.e. the level of TOM20-2 was
lower but that of TOM20-4 was higher in seedlings than in
mitochondria from cell culture. In a previous study analy-
sing the functionality of all three isoforms of TOM20, it was
observed that, in a double knock-out of tom20-2 and
tom20-3, in which TOM20-4 alone was present, import of
dual-targeted glutathione reductase was greater compared
to wild-type or any double knock-out that inactivated
TOM20-4 (i.e. tom20-3/tom20-4 or tom20-2/tom20-4) (Lister
et al., 2007). The relative abundance of a protein receptor
isoform may affect the import of a specific set of proteins,
in this instance Polc2, while others such as AtTopI may be
unaffected. Thus the relative abundance of various iso-
forms of the protein import receptors appears to differ
between tissues, and this may contribute to the ability to
detect dual targeting.
Overall, this study identified 12 new dual-targeted pro-
teins, and increased the number of dual-targeted proteins
detected in Arabidopsis to 51 (Table 2). It has identified more
proteins targeted to both mitochondria and chloroplasts,
and extended the concept of dual targeting in plant cells to
mitochondria and peroxisomes as well as mitochondria and
the nucleus.
Experimental procedures
Construction of GFP fusion proteins to analyse targeting
A Gateway� cloning cassette was constructed (Figure S3) to allowrecombination cloning of cDNA clones using Gateway� cloningtechniques according to the manufacturer’s instructions (Invitro-gen, http://www.invitrogen.com/). This strategy was chosen for allbut nine genes (see Table S2). For those nine genes, reporter geneconstructs were produced by replacing the AOX sequence in thetargeting control vectors described below with the cDNA sequenceof the gene to be tested. The Arabidopsis gene locus and theprimers used for each constructs are listed in Table S2.
Five GFP or RFP fusions were produced as controls for subcellularlocalization. The alternative oxidase (AOX) targeting signal of 42amino acids and the full-length cDNA of the small subunit of 1,5-ribulose bisphosphate carboylase/oxygenase (Rubisco SSU) werefused to GFP and RFP as mitochondrial and chloroplast controls,respectively (Carrie et al., 2007). The peroxisomal-targeting controlcontained the PTS-1 targeting signal of pumpkin (Cucurbita sp.)malate synthase fused to the C-terminus of RFP (Carrie et al., 2007;Pracharoenwattana et al., 2005).
1136 Chris Carrie et al.
ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 1128–1139
Determination of the targeting ability of GFP/RFP
fusion proteins
Biolistic transformations of GFP and RFP constructs wereperformed on Arabidopsis cell culture, seedlings and onionepidermal cells as previously reported (Carrie et al., 2007; Thir-kettle-Watts et al., 2003). The GFP and RFP plasmids (5 lg each)were co-precipitated onto gold particles and transformed usingthe biolistic PDS-1000/He system (Bio-Rad, http://www.bio-rad.-com/). Particles were bombarded onto either 2 ml of Arabidopsiscell suspension resting on filter paper on osmoticum plates, 7- to14-day-old Arabidopsis seedlings placed on filter paper on stan-dard MS plates, or onion cell epidermal peels placed on filterpaper on standard MS plates. After bombardment, all Arabidopsiscell suspension, seedlings and onion cells were placed in the darkat 22�C for 24–48 h before visualization of GFP/RFP. Localization oftransient GFP and RFP expression was performed using anOlympus BX61 fluorescence microscope (http://www.olympusmicro.com) with excitation wavelengths of 460/480 nm (GFP) and 535/555 nm (RFP), and emission wavelengths of 495–540 nm (GFP)and 570–625 nm (RFP). Subsequent images were captured usingCell� imaging software as previously described (Carrie et al.,2007; Murcha et al., 2007).
Prediction of dual-targeted proteins
Dual-targeted proteins were predicted in a number of differentways. The Predator prediction program can be used in ‘animal’ or‘plant’ mode (Small et al., 2004). The entire predicted Arabidopsisproteome was analysed twice to produce a mitochondrial-targetingprediction in the animal mode, and a plastid-targeting prediction inthe plant mode. The 803 proteins that passed both screens wereranked using the score for targeting to mitochondria using theanimal mode minus the score for mitochondrial targeting in theplant mode (Table S1a).
To predict proteins that are dual-targeted between mitochon-dria and peroxisomes, the list of predicted peroxisomal proteinsfrom Araperox (Reumann et al., 2004) was compared to knownmitochondrial proteins using the SUBA database (Heazlewoodet al., 2007) (Table S1b,c). The subcellular localizations of theoverlapping list of proteins were predicted using 10 predictors ofsubcellular localization: Target P (Emanuelsson et al., 2000, 2007),Predotar (Small et al., 2004), MitoprotII (Claros and Vincens, 1996),iPSORT (Bannai et al., 2002), Subloc (Hua and Sun, 2001),Mitopred (Guda et al., 2004), Wolfpsort (Horton et al., 2007),Multiloc (Hoglund et al., 2006), Loctree (Nair and Rost, 2005) andPeroxP (Emanuelsson et al., 2003). Proteins were selected ascandidates if either a mitochondrial location had already beenpublished or a protein showed strong mitochondrial prediction byat least five predictors.
Nuclear loci encoding proteins potentially involved inmitochondrial DNA maintenance replication and transcription(Table S1e) were identified from computational screens of theArabidopsis genome using publicly available databases at NCBI(National Centre for Biotechnology Information), TAIR (TheArabidopsis Information Resource) and AMPDB (ArabidopsisMitochondrial Protein Database) (Heazlewood et al., 2007). Thesescreens were particularly directed at identifying homologues ofestablished fungal and animal mitochondrial DNA-associatedproteins (BLAST screens at NCBI), mitochondrial homologues ofproteins reported to associate with the plastid genome (BLAST),and genes co-expressed with Arabidopsis c-type DNA polymeras-es and predicted to be involved in DNA-related processes [ATTED-II (Obayashi et al., 2007) and Expression Angler (Toufighi et al.,
2005)]. The identified loci were screened for previously uncharac-terized putative mitochondrial and plastidial gene products usingSUBA (Heazlewood et al., 2007).
Additionally, 11 transcription factors previously predicted tobe targeted to mitochondria and the nucleus (Schwackeet al., 2007) were tested for mitochondrial-targeting ability(Table S1d).
Significance of predictions
We suspected that the number of dual-targeted proteins found inthe list produced by the Predotar method (15 from 803 proteins)was greater than expected by chance. The very low proportion ofknown dual-targeted proteins among proteins predicted to beeither mitochondrial or plastidial (31 in 4320) prevents standardstatistical techniques being used to demonstrate this, so empiricalP-values were calculated by taking 10 000 random sets of 803proteins from the set of 4320 proteins predicted to be mito-chondrial or plastidial and identifying the number of known dual-targeted proteins in each of these. The P-values were taken as thenumber of random sets with at least 15 dual-targeted proteinsdivided by 10 000. Three sets of proteins contained 15 or moredual-targeted proteins, so there is an enrichment of dual-targetedproteins in the set produced by the Predotar method with aP-value = 0.0003. Similarly, empirical P-values were calculated todemonstrate the enrichment of dual-targeted proteins in the‘Mass spec’ set. In this case, 10 000 random sets of 97 proteinswere selected from the 2913 proteins with experimental evidencefor localization in the mitochondria or plastids according to theSUBA database (http://www.plantenergy.uwa.edu.au/applications/suba2/). The P-value was taken as the number of randomsets with at least three dual-targeted proteins divided by 10 000(Table 1).
In vitro import assays and Western blot analysis
In vitro import of proteins into isolated mitochondria and Westernblot analysis ere performed as previously described (Lister et al.,2007). To generate the TOM40 antibody, a recombinant proteincontaining the first 200 amino acids from the N-terminus of TOM40-1 (At3g20000) fused to an N-terminal 6· His affinity purificationtag was expressed in Escherichia coli strain BL21(DE3). Therecombinant protein was purified by denaturing Immobilized metalaffinity chromatography (IMAC), using the Bio-Rad ProfiniaTM pro-tein purification system. The resultant eluate was separated by 12%v/v SDS–PAGE, and the recombinant protein was extracted using aBio-Rad Model 422 electro-eluter. Buffer exchange was performedusing an Amicon Ultracel-5 k centrifugal filter device (http://www.millipore.com) such that the antigen was re-suspended in PBSsolution, recovering a total of 3 mg of protein for inoculation. Fourseparate doses were administered to a rabbit at regular intervalsover a 3-month period using standard protocols and Freud’scomplete adjuvant (Cooper and Paterson, 2008).
Supporting Information
Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Targeting ability of all proteins analysed in this study,except those shown in Figures 1, 2, 3 and 5.Figure S2. Proteins found to be dual-targeted to mitochondria andchloroplasts.
Dual targeting of proteins in Arabidopsis thaliana 1137
ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 1128–1139
Figure S3. Plasmid maps of the two GFP gateway destinationvectors used in this study.Table S1. List of proteins predicted to be dual-targeted usingvarious approaches as outlined in the text.Table S2. List of chromosomal loci investigated and primers used inthis study.Please note: Wiley-Blackwell are not responsible for the content orfunctionality of any supporting materials supplied by the authors.Any queries (other than missing material) should be directed to thecorresponding author for the article.
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Dual targeting of proteins in Arabidopsis thaliana 1139
ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 1128–1139
Chapter 4 Arabidopsis NAD(P)H dehydrogenases are dual targeted
54
Chapter 4
Arabidopsis NAD(P)H dehydrogenases are dual targeted
Chapter 4 Arabidopsis NAD(P)H dehydrogenases are dual targeted
55
Foreword to Study III Upon closer examination of the list of candidate dual targeted proteins defined
in study II, it was observed that a number of type II NAD(P)H dehydrogenases (ND)
were predicted to target both mitochondria and peroxisomes. Typically, ND proteins
have been located on the mitochondrial inner membrane where they can oxidise
NAD(P)H (Michalecka et al., 2003; Rasmusson et al., 2004; Elhafez et al., 2006). In
Arabidopsis there are seven genes, that encode for ND proteins, three have been defined
as external (NDB1, 2 and 4) and three have been defined as internal (NDA1 and 2 and
NDC1) (Michalecka et al., 2003; Elhafez et al., 2006) whilist the remaining gene,
NDB3, is thought to be a pseudogene (Elhafez et al., 2006). Over the past decade a
number of different studies have defined ND proteins as mitochondrial using GFP
tagging (Michalecka et al., 2003), in vitro uptake experiments (Elhafez et al., 2006),
Western blotting (Rasmusson and Agius, 2001), and enzymatic activity assays
(Svensson and Rasmusson, 2001; Geisler et al., 2004). However, when looking for
proteins targeted to mitochondria and peroxisomes, a number of ND proteins were
predicted to contain not only mitochondrial targeting signals but also PTS1 like signals
at their C-terminus (NDA1 and 2 and NDB1). During this analysis it was also predicted
that NDC1 was a plastid protein, which is not surprising considering its cyanobacterial
ancestry (Michalecka et al., 2003).
Using the established techniques from study II, GFP fusions were made of all
Arabidopsis ND proteins at both the N and C-terminus. It was found that NDA1 and 2
and NDB1 were dual targeted to mitochondria and peroxisomes by two separate
targeting signals: an N-terminal targeting signal for mitochondria, and a C-terminal
PTS1 for peroxisomal targeting. This second signal had previously been missed, due to
studies only using the N-terminal part of the protein for targeting studies. It was also
discovered that NDC1 was targeted to both mitochondria and plastids, using GFP
tagging and in vitro import experiments. It is proposed that the reason why the plastid
targeting was missed in previous studies was because only the N-terminal part of the
protein was used (Michalecka et al., 2003), not the full length protein as was used in
this study. The dual targeting of ND proteins raises interesting questions as to their roles
within plant metablolism.
FEBS Letters 582 (2008) 3073–3079
Type II NAD(P)H dehydrogenases are targeted to mitochondriaand chloroplasts or peroxisomes in Arabidopsis thaliana
Chris Carriea, Monika W. Murchaa, Kristina Kuehna, Owen Duncana,Michelle Barthetb, Penelope M. Smithb, Holger Eubela, Etienne Meyera,
David A. Dayb, A. Harvey Millara, James Whelana,*
a ARC Centre of Excellence in Plant Energy Biology, MCS Building M316 University of Western Australia,35 Stirling Highway, Crawley 6009, Western Australia, Australia
b ARC Centre of Excellence in Plant Energy Biology, School of Biological Sciences, University of Sydney, NSW, Australia
Received 21 July 2008; revised 30 July 2008; accepted 31 July 2008
Available online 12 August 2008
Edited by Ulf-Ingo Flugge
Abstract We found that four type II NAD(P)H dehydrogen-ases (ND) in Arabidopsis are targeted to two locations in thecell; NDC1 was targeted to mitochondria and chloroplasts, whileNDA1, NDA2 and NDB1 were targeted to mitochondria andperoxisomes. Targeting of NDC1 to chloroplasts as well as mito-chondria was shown using in vitro and in vivo uptake assays anddual targeting of NDC1 to plastids relies on regions in the ma-ture part of the protein. Accumulation of NDA type dehydrogen-ases to peroxisomes and mitochondria was confirmed usingWestern blot analysis on highly purified organelle fractions. Tar-geting of ND proteins to mitochondria and peroxisomes isachieved by two separate signals, a C-terminal signal for peroxi-somes and an N-terminal signal for mitochondria.� 2008 Federation of European Biochemical Societies. Pub-lished by Elsevier B.V. All rights reserved.
Keywords: Chloroplast; Mitochondria; Peroxisome; Dualtargeting; Green fluorescent protein; Alternative NAD(P)Hdehydrogenase
1. Introduction
A hallmark of eukaryotic cells is the partitioning of various
biochemical pathways out of the cytosolic milieu and into dis-
crete organelles. Although the compartmentalisation of vari-
ous biochemical functions allows specialisation, it requires
that many functions are duplicated and thus many enzymatic
activities take place in more than one organelle. In the majority
of cases these common functions are performed by different
proteins, encoded by distinct genes, that are each targeted to
a single location in the cell [1]. However, in other cases it ap-
pears that the same function in different organelles is carried
out by the same protein that is targeted to two locations, a
Abbreviations: AOX, alternative oxidase; GFP, green fluorescentprotein; KAT2, 3-ketoacyl-CoA thiolase; ND, type II alternativeNAD(P)H dehydrogenase; RFP, red fluorescent protein; SSU, Rubi-sco small subunit of ribulose 1,5 bisphosphate carboxylase/oxygenase;TIM17-2, translocase of the inner mitochondrial membrane
*Corresponding author. Fax: +61 8 93801148.E-mail address: [email protected] (James Whelan).
0014-5793/$34.00 � 2008 Federation of European Biochemical Societies. Pu
doi:10.1016/j.febslet.2008.07.061
process called dual targeting. This was first reported for gluta-
thione reductase from pea, which is targeted to both mitochon-
dria and chloroplasts [2]. To date, studies in several plants
suggest that more than 30 proteins are dual targeted to mito-
chondria and chloroplasts [3].
The targeting of proteins is routinely assessed by attaching a
reporter, most often green fluorescent protein (GFP), to the
protein being studied and the intra-cellular distribution of fluo-
rescence measured [4]. This approach is convenient and sensi-
tive and has been used widely to define dual targeting to
mitochondria and chloroplasts [5–8]. However, this approach
has some limitations that depend on the nature of the con-
structs. Firstly, for proteins that may be targeted to two loca-
tions using two signals in different parts of the protein
sequence, GFP fusion to one part of the protein can mask
an adjacent signal – resulting in localisation to only one of
its in vivo destinations. Secondly, targeting ability can be af-
fected by the nature of the passenger protein. This occurs even
for proteins targeted to a single location [9,10], but it seems to
be even more pronounced for dual targeted proteins. In two
independent studies examining the role of the mature protein
for dual targeted proteins to mitochondria and chloroplasts,
both concluded that the passenger or mature protein influ-
enced dual targeting ability [11,12].
Type II NAD(P)H dehydrogenases are typically located on
the mitochondrial inner membrane where they can oxidise
NAD(P)H and are insensitive to the complex I inhibitor rote-
none [13–15]. Seven genes encode putative type II NAD(P)H
dehydrogenases in Arabidopsis, three have been defined as
external (NDB 1, 2 and 4) and three defined as internal
NAD(P)H dehydrogenases (NDA 1 and 2 and NDC1)
[13,14]. The remaining gene encoding a putative external
NAD(P)H dehydrogenase, NDB3, could not be cloned by a
number of groups and thus is either a pseudogene or its expres-
sion is very restricted [13,16]. Previous studies using GFP tag-
ging have shown NDA1, NDA2, NDB1, NDB2 and NDC1 to
be targeted to mitochondria [14], in vitro mitochondrial uptake
assays have shown NDA1, NDA2, NDB1, NDB2, NDB4 and
NDC1 to be imported into mitochondria [13], and a number of
studies using Western blot analysis of mitochondrial proteins
and/or cellular fraction with antibodies raised against peptides
from potato NDA1 and NDB1 have all concluded a mitochon-
drial localisation for these proteins [17–19]. Additionally over
two decades of biochemical analysis have shown that the
blished by Elsevier B.V. All rights reserved.
3074 C. Carrie et al. / FEBS Letters 582 (2008) 3073–3079
activities associated with these proteins are located in mito-
chondria [15,20]. Thus it can be concluded that these proteins
are located in mitochondria.
However the set of proteins predicted to be located in per-
oxisomes by the AraPerox database, with medium to high con-
fidence, identifies three of these mitochondrial type II
NAD(P)H dehydrogenases [21]. Thus we re-assessed the tar-
geting ability of all six NAD(P)H dehydrogenases with the
view that they may also be located in other cellular organelles
in addition to mitochondria.
2. Materials and methods
2.1. Sequence analysis and cloningThe full length coding sequences of NDA1, NDA2, NDB1, NDB2,
NDB4 and NDC1 were cloned as both N- and C-terminal GFP fusionsby Gateway cloning under the control of the 35S CaMV promoter.Additionally the last 10 amino acids of NDA1, NDA2, NDB1 andNDB2 were cloned to the C-terminus of GFP. The alternative oxidase(AOX) targeting signal, the full length targeting sequence of small sub-unit of 1,5 ribulose bisphosphate carboxylase/oxygenase (SSU Rubi-sco) and the peroxisomal targeting signal SRL of pumpkin malatesynthase, were fused to red fluorescent protein (RFP) and used asmitochondrial, chloroplast and peroxisomal controls, respectively[22–24].
The constructs were used to transform Arabidopsis suspension cul-ture cells, Arabidopsis seedlings (1–2 weeks old) and onion epidermalcells by biolistic transformation as previously outlined [25]. Fluores-
Physcomitrella NDB1 RPhyscomitrella NDB2 SPhyscomitrella NDB3 S
Arabidopsis NDB1 SRI Potato NDB1 SRI
Grape NDB1 SRIRice NDB1 SRIArabidopsis NDB2 SSI
Grape NDB2 SRIGrape NDB3 SRI
Arabidopsis NDB4 SSIRice NDB2 SSL
Rice NDB3 LCS
Fig. 1. ClustalW alignment of type II NAD(P)H dehydrogenases from a vaNAD(P)H dehydrogenases from a variety of plants revealed that several coterminal end of the protein. The predicted strength of the PTSI signal wamitochondria and/or chloroplasts is shown. Arabidopsis thaliana NDAt2g2990;NP_180560, NDB1 At4g28220;NP_567801, NDB2 At4g05020;NCAB52796, NDB1 CAB52797, Populus trichocarpa NDA1 ABK95883, VitNDB1 CAO41235, NDB2 CAO16606, NDB3 CAO41237, Saccharomyces cersativa NDC1 Os06g11140:BAD35311, NDA1 Os01g61410:NP_915326.1, NDOs05g26660:AAV43826, NDB3 Os08g04630:XP_480031.1, ChlamydomonXP_001702271, NDB1 XP_001703643, Physcomitrella patens NDC1 manNDA1 manually annotated from scaffold 28 of Physcomitrella genome [39],NDB3 XP_001764062. Targeting prediction for all proteins are shown in Su
cence patterns were obtained 24 h after transformation by visualizationunder an Olympus BX61 fluorescence microscope, with excitationwavelengths of 460–480(GFP) and 535–555(RFP). Emissions were col-lected for GFP between 495 and 540 and RFP between 570 and 625,and imaged using the CellR imaging software. To ensure no cross overin detection of signals AOX-RFP and SSU-GFP were co-transformedto ensure that the filters were detecting the appropriate signal.
2.2. Determination of subcellular targeting abilityN- and C-terminal GFP-tagged proteins were used to transform
Arabidopsis cell suspension culture, 1–2 week old Arabidopsis seed-lings and onion epidermal cells by biolistic transformation as previ-ously outlined [25]. For each construct to be tested threetransformations were carried out, the test construct with a mitochon-drial, plastidic and peroxisomal control. In vitro import assays intoisolated Arabidopsis mitochondria and pea chloroplasts were carriedout as previously outlined [23,25].
2.3. Antibody production and Western blottingAntibodies were raised in rabbit against NDA1, amino acids 57–236
and the NDB2 specific peptide at amino acids 438–452(ETDDVSKNNIELKIE). The specificity of the antibodies was testedagainst recombinant proteins synthesised in a wheat germ translationlysate according to manufacturers instructions (Roche, Sydney), pro-grammed to synthesise NDA1, NDA2 and NDB2 by making lineartemplates by PCR as per manufactures instructions (Roche, Sydney).
Mitochondria and peroxisomes were purified from 7 day old cell sus-pension culture using free flow electrophoresis as described by Eubel etal. [26]. Western blot analysis was carried out against 20 lg of mito-chondrial and peroxisomal proteins separated by SDS–PAGE aspreviously outlined [27].
Grape NDC1Rice NDC1
Physcomitrella NDC1Chlamydomonas NDC1
Arabidopsis NDA1 SRIArabidopsis NDA2 SRI
Poplar NDA1 SRIGrape NDA1 SRIPotato NDA1 SRIRice NDA1 SRI
Grape NDA2 RIGRice NDA2 RIG
Physcomitrella NDA1 SRFPhyscomitrella NDA2 SRF
Chlamydomonas NDA1 SRWChlamydomonas NDA2 SLF
Yeast NDI KGLYeast NDE1 SSIYeast NDE2 SSV
Chlamydomonas NDB1 SRVVERMRM
Arabidopsis NDC1
Strong PTS-1Moderate PTS-1Weak PTS-1MitochondrialPlastid
riety of plants and yeast. Alignment of the sequences encoding type IIntained putative peroxisomal type I targeting signals (PTSI) at the C-s taken from AraPerox [21]. The predicted ability to be targeted toC1 At5g08740;NP_568205, NDA1 At1g07180;NP_563783, NDA2P_180560, NDB4 At2g20800;NP_179673, Solanum tuberosum NDA1is vinifera NDA1 CAO21440, NDC1 CAO71655, NDA2 CAO67571,evisiae NDI NP_013586, NDE1 NP_013865, NDE2 NP_010198, OryzaA2 Os07g377730:NP911221.1, NDB1 Os06g47000:BAD45556, NDB2as reinhardtii NDC1 ABR53723, NDA1 XP_001698901, NDA2ually annotated from scaffold 101 of Physcomitrella genome [39],NDA2 XP_001769969, NDB1 XP_001766162, NDB2 XP_001759207,pplementary Table 1.
C. Carrie et al. / FEBS Letters 582 (2008) 3073–3079 3075
3. Results
A ClustalW alignment of all ND sequences available from
various plant species and yeast revealed the amino acid se-
quence SRI at the C-terminal end of Arabidopsis NDA1,
NDA2 and NDB1 (and NDB3), and in a variety of ND pro-
teins from other plants. Other PTS I type targeting signals,
most notably SRM or SSI, were also found in ND sequences
[28] (Fig. 1). The fact that these amino acids are not present
in all ND sequences suggests that this tripeptide is not required
for function, opening the possibility that it may play a role in
defining subcellular localisation via its peroxisomal targeting
activity [15]. Analysis of NDC1 sequences from Arabidopsis,
rice and Chlamydomonas reinhardtii predicted plastid-targeting
in all three species based on the N-terminal region (Supple-
mentary Table 1), even though these proteins display very
low levels of sequence identity in this region (data not shown).
Examination of the gene constructs used in a previous study
that indicated an exclusive mitochondrial localisation for these
proteins, revealed that only the N-terminal region was used in
the GFP fusions [14], amino acids 1–55 for NDA1, 1–60 for
NDA2, 1–59 for NDB1 and NDB2 and amino acids 1–83
for NDC1.
3.1. NDC1 is targeted to mitochondria and chloroplasts
The full-length cDNA for NDC1 was placed in front of GFP
and its subcellular localisation examined by particle bombard-
ment. As controls, the cells transformed with the NDC1-GFP
construct were co-transformed either with plastid targeted
RFP using the targeting signal of the small subunit of 1,5
Fig. 2. Subcellular targeting of NDC1 using GFP tagging (A) In vitro uptakframe with GFP and co-transformed into Arabidopsis cells with mitochonpanel). (B) In vitro uptake of NDC1 into isolated mitochondria and chmitochondria (lane 2) and chloroplasts (lane 3) under conditions that suppassessed by insensitivity to added protease. Both organelles processed themitochondria, uptake was sensitive to the addition of valinomycin (lanes 5 an7 and 8). (C) The specificity of import of protein into isolated organellesoxygenase (SSU) that was only imported into chloroplasts and alternative o
ribulose bisphosphate carboxylase/oxygenase (SSU Rubisco-
RFP) or the mitochondrial alternative oxidase targeting signal
(AOX-RFP). Targeting of NDC1-GFP to chloroplasts was
clearly observed in Arabidopsis suspension cells ( Fig. 2A),
the pattern was clearly not identical to AOX-RFP but resem-
bled that of SSU-RFP quite closely. This is in contrast to what
has been previously reported where a mitochondrial localisa-
tion was concluded when the first 83 amino acids of NDC1
was used [14]. However we routinely observed a weaker signal,
similar to the pattern obtained with AOX-RFP. Thus we
tested the targeting ability in a variety of tissues, namely Ara-
bidopsis seedlings and onion epidermal cells. Transformation
of these tissues resulted in the detection of two distinct signals,
a plastid signal evidenced by relatively large organelles, 2–
4 lM in diameter and few in number and smaller organelles,
1 or less lM in diameter typical of a mitochondrial pattern.
The mitochondrial targeting ability of NDC1 that we observed
in this study is consistent with previous results using GFP and
in vitro uptake assays [13,14].
To confirm that NDC1 could target to both chloroplasts and
mitochondria, in vitro uptake assays with isolated Arabidopsis
mitochondria and pea chloroplasts were carried out. Upon
incubation with isolated chloroplasts and mitochondria the
NDC1 precursor protein with a mol mass of 70 kDa was im-
ported into a protease resistant location and processed to a
mature size with a mol mass of 60 kDa (Fig. 2B, lanes 1–3).
Both organelles appeared to process the precursor protein to
the same mature protein, to confirm this import reactions into
mitochondria and chloroplasts were loaded into the same lane
to determine any small difference in mobility, none was
e assays (B and C). A) The full-length cDNA for NDC1 was fused indrial targeted RFP (top panel) or chloroplast targeted RFP (bottomloroplasts. Precursor proteins (lane 1) were incubated with isolatedort the uptake of proteins into the respective organelles. Uptake wasprecursor to a mature protein with the same mobility (lane 4). Ford 6). For chloroplasts uptake was inhibited by addition of CuCl2 (laneswas confirmed using the precursor of 1,5 bisphosphate carboxylase
xidase (AOX) that was only imported into mitochondria.
3076 C. Carrie et al. / FEBS Letters 582 (2008) 3073–3079
detected (Fig. 2B, lane 4). As the translation of the precursor
alone also produces a protein with a mol mass of 60 kDa,
likely due to translation initiation at an internal methionine,
such as amino acid 47 in NDC1. Translation initiation at
Fig. 3. Subcellular targeting of NDA1, NDA2, NDB1 and NDB2. GFP wastargeting assessed by particle bombardment of Arabidopsis suspension cellsmitochondrial targeted RFP or peroxisomal targeted RFP as controls. (A) SNDB2-GFP. (B) Subcelluar targeting pattern obtained with NDA1, NDA2amino acids used is shown for each construct.
internal methionine residues is frequently observed with in vi-
tro translation lysates [27]. Thus we confirmed that the prote-
ase resistance was due to import into the respective organelle.
Import into mitochondria was inhibited by the addition of
fused to the different proteins at the N- or C-terminal and subcellular, 1–2 weeks old Arabidopsis seedlings and onion epidermal cells withubcelluar targeting pattern obtained with AOX-GFP, KAT2-GFP andand NDB1 fused to GFP. The position of the GFP and the number of
C. Carrie et al. / FEBS Letters 582 (2008) 3073–3079 3077
valinomycin (Fig. 2B, lanes 5 and 6) [27], and import into chlo-
roplast inhibited by the addition of CuCl2 (Fig. 2B, lanes 7 and
8) [29]. The specificity of import into the respective organelles
was confirmed as the small subunit of 1,5 bisphosphate (SSU)
was only imported into chloroplasts and the alternative oxi-
dase precursor only imported into mitochondria (Fig. 2C, left
panel).
3.2. NDA1, NDA2 and NDB1 are targeted to mitochondria and
peroxisomes
In order to determine the localisation of the other ND pro-
teins, N- and C-terminal GFP fusions were made followed by
particle bombardment. To determine a mitochondrial and per-
oxisomal pattern chimeric constructs with the AOX and KAT2
linked to GFP were used (Fig. 3A, image series 1 and 2). In the
case of NDB2 attaching GFP to the C-terminal resulted in tar-
geting to mitochondria as evidenced by co-localisation with
AOX-RFP (Fig. 3A, images 3a–3c). Attaching the last 10 ami-
no acids of NDB2 to the C-terminal end of GFP resulted in a
cytosolic localisation for GFP, as evidenced by fluorescence
throughout the cell, in all tissues tested (Fig. 3A, image series
4). In contrast when NDA1, NDA2 and NDB1 were tested in a
similar manner both mitochondrial and peroxisomal targeting
ability was detected. C-terminal fusions gave an exclusively
Anti NDA1
Anti NDB2
Anti TIM17-2
Anti Kat2
Mit
A1 B2---A
Anti 6-His
Anti A1
Anti B2
60 kDa
62 kDa
C
Fig. 4. Western blot analysis of mitochondrial and peroxisomal fractions prantibodies. Wheat germ lysate (20 lg) programmed to synthesise each of themembrane and probed with antibodies raised against NDA1 and NDB2 totranslation lysate programmed to synthesis b-glucuronidase (GUS), NDA1 aflow electrophoresis were separated by SDS–PAGE, blotted to a nitrocelluloused is indicated to the right of the panel and the apparent mol mass of the cand NDA2 the precursor size of the protein is detected when probing in vitrowhen probing organelle fractions.
mitochondrial localisation, based on co-localisation with
AOX-RFP (Fig. 3B, images 1a–1c, 3a–3c and 5a–5c). This is
consistent with the mitochondrial targeting ability previously
observed with these proteins [14]. However when the last 10
amino acids of NDA1, NDA2 and NDB1 were placed at the
C-terminal region of GFP peroxisomal targeting was observed
(Fig. 3B, images 2d–2f, 4d–4f and 6d–6f). The peroxisomal tar-
geting ability of these constructs was also detected in Arabid-
opsis seedlings and onion cells (Fig. 3B, images 2g and 2h, 4g
and 4h and 6g and 6h). Thus we concluded that these proteins
were targeted to peroxisomes in addition to mitochondria.
NDB4 targeted GFP to mitochondria as previously reported
(Supplementary Fig. 1) [14].
To confirm the dual location of NDA1 in mitochondria and
peroxisomes we raised antibodies against NDA1, expected to
be located in both locations from results above, and NDB2,
expected to be located only in mitochondria from results
above. We confirmed that the NDA1 and NDB2 antibodies
did not cross react with the other antigen by over-expression
of the respective proteins in an in vitro translation lysate prob-
ing with Anti 6-His antibodies that detected both proteins,
Anti A1 antibodies that detected only NDA1 and Anti B2 anti-
bodies that detected only B2 (Fig. 4A). As the NDA1 antibody
was raised against a fragment of the NDA1 protein on 180
55 kDa
62 kDa
32 kDa
44 kDa
Per
Anti A1
B
Anti 6-His
GUS A1 A2
60 kDa
68 kDa
59 kDa
60 kDa
obed with various antibodies. (A) Confirmation of NDA1 and NDB2ND proteins was separated by SDS–PAGE, blotted to a nitrocelluloseconfirm that they detected their target antigens. (B) As A except thatnd NDA2. (C) 20 lg of mitochondria or peroxisomes purified by freese membrane and probed with antibodies as indicated. The antibodyross reacting protein indicated in the left in kDa. Note that for NDA1
synthesised protein whereas the mature size of the protein is detected
3078 C. Carrie et al. / FEBS Letters 582 (2008) 3073–3079
amino acids that displayed 83% sequence identity with the cor-
responding region of NDA2 we tested if the NDA1 antibody
cross reacted with NDA2. No cross reactivity was detected
with full length in vitro synthesised NDA2 (Fig. 4B).
Highly purified mitochondria and peroxisome fractions were
isolated from Arabidopsis cells [26] and proteins separated by
SDS–PAGE and subjected to Western blotting. In control
experiments, we used antibodies against proven markers of
mitochondria (TIM17-2 (Translocase of the Inner Mitochon-
drial membrane; [27]), and peroxisomes (KAT2 (3-ketoacyl-
CoA thiolase; [22,30]). These antibodies reacted strongly with
mitochondrial and peroxisomal fractions, respectively, and
much more weakly with the other fraction, indicating a small
degree of cross-contamination between the fractions (Fig.4B).
Densitometric analysis revealed the KAT2 signal in mitochon-
dria was �5% of that detected in peroxisomes, whilst the
TIM17-2 signal in peroxisomes was �1–2% of the signal that
could be detected in mitochondria (when the blot was overex-
posed).
Probing with antibodies raised against NDA1 resulted in the
detection of a single protein band with an apparent molecular
mass of 55 kDa, in both mitochondrial and peroxisome frac-
tions ( Fig. 4B). The blots indicated that there was more
NDA1 protein in the peroxisomal fraction than in the mito-
chondrial one, confirming that these proteins are found in both
compartments. Probing mitochondrial and peroxisomal frac-
tions with antibodies raised against the NDB2 specific peptide
produced a band only in the mitochondrial fraction (Fig. 4B),
confirming that it can target to mitochondria but not to per-
oxisomes. Importantly, this latter result also shows that the
very small amount of cross-contamination between the two
isolated fractions cannot explain the dual localisation of the
NDA1 signal. Thus the Western blot results confirm the
GFP data.
4. Discussion
In this study we have shown that four ND proteins, NDA1,
NDA2, NDB1 and NDC1 are dual targeted. The dual target-
ing ability of ND proteins was overlooked in previous GFP
studies due to a number of technical parameters, namely the
nature of the GFP-protein constructs used in each study. In
the case of NDC1, it appears that the mature protein sequence
is required for its dual localisation by GFP (Fig. 2), as ob-
served for other dual targeted proteins [12,31]. The dual target-
ing of NDA1, NDA2 and NDB1 to mitochondria and
peroxisomes is dictated by two distinct signals. In the case of
the NDAs, the apparent Mr of the mature protein observed
in peroxisomes and mitochondria was identical (Fig. 4C). As
NDA proteins are processed upon import into mitochondria
[13], this strongly suggests that they are also processed upon
import into peroxisomes. It has been shown previously that
peroxisomes recognise N-terminal PTS2 type targeting signals
that are removed upon import and the processing of the NDA
proteins could be carried out by the same peptidase as both
NDA1 and NDA2 have a cysteine residue at amino acids 35
and 38, respectively, which defines the processing site by this
peptidase [32]. Alternatively, the NDA proteins may be pro-
cessed by pitrilysin-like metallopeptidase present in peroxi-
somes [32]. These enzymes belong to the same family of
proteases as the mitochondrial processing peptidase [33].
The mitochondrial pattern obtained with GFP with NDA1,
NDA2 and NDB1 differed slightly to that obtained from
AOX-RFP. Close examination of the merged images revealed
that the GFP fluorescence appeared at the periphery of the
mitochondrion, thus the GFP and RFP fluorescence co-local-
ise, but are not identical. A similar pattern of GFP fluores-
cence is routinely obtained when using outer membrane
mitochondrial proteins in humans and Arabidopsis [34,35].
This pattern may be due to the fact that GFP attached to
the C-terminal of an inner membrane protein will not be
�pulled� into mitochondria. The C-terminal of the ND proteins
may be located in the intermembrane space and thus never en-
ter the mitochondrial matrix. Thus the GFP attached to the C-
terminal end of these proteins remains outside the mitochon-
drion. Using only the N-terminal predicted targeting region re-
sults in a typical mitochondrial pattern as previously observed
[14], as the default targeting information for mitochondria dic-
tated a matrix location [36]. Secondary signals dictate the in-
tra-organelle location and topology of proteins, such as
transmembrane regions and the location of positive residues
relative to transmembrane regions [36].
The cellular role of various ND proteins now needs to be
re-evaluated in light of their dual localisation. For instance
NDC1 gene expression is enhanced by light treatments [16]
but the protein is also known to be halved in abundance in
plastoglobules during high light treatment [37]. So what im-
pact does this transcriptional light response have on the mito-
chondrial pool of NDC1 protein? Likewise, Western blot
analysis with potato mitochondria revealed changes in
NDA protein in a diurnal manner [18], and it is now unclear
how much of this may be attributed to a mitochondrial func-
tion as opposed to a peroxisomal function, or differential
contamination of mitochondria with peroxisomes. Further,
loss of or over-expression of potato NDB1 has been shown
to alter NADPH/NADH ratio in cells [38], but this may be
related to its activity in peroxisomes rather than mitochon-
dria.
Seven genes encode alternative ND proteins in Arabidopsis,
two NDA like proteins, four NDB type proteins and a single
NDC type protein, the latter proposed to be derived from
the cyanobacterial ancestor that gave rise to the plastid endo-
symbiosis [14]. It is tempting to speculate from the prediction
of targeting ability of these proteins from a variety of plants
( Fig. 1, Supplementary Table 1) that genes encoding single
NDA and NDB type proteins underwent duplication followed
by acquisition of additional targeting signals by some proteins.
In the case of NDC it may have acquired dual targeting ability
upon transfer of the gene from the organelle to the nucleus, or
alternatively a location specific signal subsequently acquired
dual targeting ability over time.
Acknowledgements: This work was supported by an AustralianResearch Council Grant DP0664692, ARC Australian PostdoctoralFellowships to M.W.M. and H.E., and an ARC AustralianProfessorial Fellowship to A.H.M.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.febslet.2008.
07.061.
C. Carrie et al. / FEBS Letters 582 (2008) 3073–3079 3079
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Chapter 5 Plant mitochondrial protein import receptors
62
Chapter 5
Plant mitochondrial protein import receptors
Chapter 5 Plant mitochondrial protein import receptors
63
Foreword to Study IV Studies I, II and III identified new dual targeted proteins in Arabidopsis. Study
IV, therefore aimed to identify how these dual targeted proteins are imported into
Arabidopsis mitochondria, by investigating the mitochondrial outer membrane protein
import receptors. While this study contains work on all the putative import receptors
(Tom20, Metaxin and OM64), only the work regarding OM64 is being presented for
this thesis, the work carried out on Tom20 and Metaxin was not part of this thesis (see
declaration form).
Using genome searches and biochemical purification of the plant TOM complex
there has been no evidence found for a Tom70 orthologue in plants (Jansch et al., 1998;
Chan et al., 2006). However, it was discovered that Arabidopsis mitochondria contain a
protein anchored to the outer membrane with a large cytosolic domain termed OM64
(Chew et al., 2004). Interestingly, it was seen that OM64 contains three TPR motifs and
is related to the Toc64 protein from plastids (Chew et al., 2004). Toc64 is an outer
envelope protein of plastids and is thought to be involved in protein import into plastids
(Qbadou et al., 2006). To date, no functional role for OM64 has been demonstrated,
however it has been proposed that OM64 may play a similar role to Tom70 in yeast
(Chew et al., 2004). Thus OM64, was of interest as it is related to an import component
of plastids and is found on the outer mitochondrial membrane. Study IV aimed to test
the hypothesis that OM64 is involved in the import of dual targeted proteins into
mitochondria. However, no specific role for OM64 in the import of dual targeted
proteins was found. Insertional inactivation, in vitro competition experiments with
overexpressed OM64, antibody inhibition assays, and direct interaction assays all
suggested a role for OM64 in the import of proteins into mitochondria.
Functional Definition of Outer Membrane Proteins Involved inPreprotein Import into Mitochondria W
Ryan Lister,1 Chris Carrie,2 Owen Duncan,2 Lois H.M. Ho, Katharine A. Howell,Monika W. Murcha, and James Whelan3
Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley 6009, Western
Australia, Australia
The role of plant mitochondrial outer membrane proteins in the process of preprotein import was investigated, as some of
the principal components characterized in yeast have been shown to be absent or evolutionarily distinct in plants. Three
outer membrane proteins of Arabidopsis thaliana mitochondria were studied: TOM20 (translocase of the outer mitochon-
drial membrane), METAXIN, and mtOM64 (outer mitochondrial membrane protein of 64 kD). A single functional Arabidopsis
TOM20 gene is sufficient to produce a normal multisubunit translocase of the outer membrane complex. Simultaneous
inactivation of two of the three TOM20 genes changed the rate of import for some precursor proteins, revealing limited
isoform subfunctionalization. Inactivation of all three TOM20 genes resulted in severely reduced rates of import for some
but not all precursor proteins. The outer membrane protein METAXIN was characterized to play a role in the import of
mitochondrial precursor proteins and likely plays a role in the assembly of b-barrel proteins into the outer membrane. An
outer mitochondrial membrane protein of 64 kD (mtOM64) with high sequence similarity to a chloroplast import receptor
was shown to interact with a variety of precursor proteins. All three proteins have domains exposed to the cytosol and
interacted with a variety of precursor proteins, as determined by pull-down and yeast two-hybrid interaction assays.
Furthermore, inactivation of one resulted in protein abundance changes in the others, suggesting functional redundancy.
Thus, it is proposed that all three components directly interact with precursor proteins to participate in early stages of
mitochondrial protein import.
INTRODUCTION
The mitochondrial protein import machinery has been most com-
prehensively characterized in yeast (Saccharomyces cerevisiae),
Neurospora crassa, and to a lesser extent in mammalian sys-
tems. Hetero-oligomeric translocation complexes in the outer
and inner membranes mediate the recognition, import, and sub-
organellar sorting of mitochondrial precursor proteins (Neupert,
1997; Pfanner and Geissler, 2001; Hoogenraad et al., 2002;
Truscott et al., 2003; Wiedemann et al., 2004). The translocase of
the outer mitochondrial membrane (TOM) complex facilitates the
recognition of precursor proteins and their translocation through
the outer membrane (Taylor and Pfanner, 2004). In yeast, the
outer membrane receptors TOM20, TOM22, and TOM70 asso-
ciate with the general import pore that consists of the pore-
forming TOM40, and TOM7, TOM6, and TOM5. TOM20 and
TOM70 are N-terminal anchored primary receptor proteins that
recognize precursor proteins with N-terminal and internal tar-
geting information, respectively (Wiedemann et al., 2004). Pre-
cursor proteins subsequently interact with the TOM22 receptor,
which delivers them to the general import pore (Taylor and
Pfanner, 2004). Operating in concert with the TOM complex, the
sorting and assembly machinery (SAM) complex (also called
topogenesis of mitochondria outer membrane b-barrel proteins
[TOB]) in the outer mitochondrial membrane inserts proteins into
the outer membrane (Pfanner et al., 2004; Taylor and Pfanner,
2004; Habib et al., 2005; Paschen et al., 2005). Although initially
thought to be a protein import receptor of the TOM complex
(Gratzer et al., 1995), SAM37 (also called MAS37/TOM37) was
subsequently reported to be located in a distinct outer mem-
brane complex, SAM, and was demonstrated to function in the
sorting and assembly of b-barrel proteins (Wiedemann et al.,
2003). The SAM complex, consisting of SAM50 (TOB55), SAM35
(TOB38), SAM37, and MDM10 (TOM13), in yeast and N. crassa,
is required for the correct assembly of complex b-barrel proteins
into the outer membrane after import through the TOM complex,
but the precise molecular functions of SAM35 and SAM37 have
not yet been elucidated (Wiedemann et al., 2003; Paschen et al.,
2005; Neupert and Herrmann 2007). Proteins destined for the
inner membrane and matrix interact with two discrete TIM
(translocase of the inner mitochondrial membrane) complexes
(Wiedemann et al., 2004).
Experimental isolation of mitochondrial proteins coupled with
genome sequence analysis in animals and plants has revealed that
much of the import apparatus is conserved throughout diverse
eukaryotic lineages (Herrmann, 2003; Dyall et al., 2004; Lister
et al., 2005; Dolezal et al., 2006). However, considerable dif-
ferences exist in the TOM complex subunit composition between
1 Current address: Plant Biology Laboratory, Salk Institute for BiologicalStudies, La Jolla, CA 92037.2 These authors contributed equally to this work.3 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: James Whelan([email protected]).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.107.050534
The Plant Cell, Vol. 19: 3739–3759, November 2007, www.plantcell.org ª 2007 American Society of Plant Biologists
species; only the core translocase module of TOM40, TOM22, and
TOM7 is conserved (Dolezal et al., 2006), and the plant TOM22 has
lost the N-terminal receptor domain present in yeast and animals
(Macasev et al., 2004). Isolation of the plant TOM complex also
identified small proteins analogous to yeast TOM5 and TOM6 and
a 23-kD protein (plant TOM20) analogous to yeast/animal TOM20,
but none displayed significant protein sequence similarity to the
yeast or mammalian counterparts (Heins and Schmitz, 1996;
Jansch et al., 1998; Werhahn et al., 2003). Subsequent elucidation
of the solution structure of the plant TOM20 showed it to have a
similar tertiary structure but reverse domain arrangement to yeast/
animal TOM20 (Perry et al., 2006). Given that there is no signif-
icant sequence similarity between plant TOM20 and that of yeast
or mammals, it is proposed that convergent evolution has lead to
a receptor with the same function but reversed orientation (Lister
and Whelan, 2006; Perry et al., 2006). As plant TOM20 is not
orthologous to yeast or mammalian TOM20, it is of interest to
determine if it plays the same role in the TOM complex.
No clear plant homolog of the yeast and animal TOM70 re-
ceptor can be identified in the genomes of Arabidopsis thaliana
or rice (Oryza sativa; Chan et al., 2006), in agreement with the lack
of any biochemical evidence for this component in the purified
plant TOM complex (Jansch et al., 1998). Interestingly, anchored
in the outer membrane of Arabidopsis mitochondria is mtOM64, a
paralog of the chloroplast outer envelope protein import receptor
At TOC64-III (translocase of the outer chloroplast envelope)
(Chew et al., 2004; Qbadou et al., 2006). Notably, inactivation of
both plastid-localized TOC64 orthologs in Physcomitrella patens
and inactivation of TOC64 in Arabidopsis had no effect on protein
import into plastids (Hofmann and Theg, 2005; Aronsson et al.,
2007). To date, no functional role has been demonstrated for
mtOM64 in Arabidopsis. Plants also display differences in the
SAM complex in comparison to yeast; in plants, only SAM50 can
be clearly identified by sequence similarity (Lister et al., 2005).
Arabidopsis METAXIN was identified by sequence similarity to the
human METAXIN 1 protein; the latter displays limited sequence
similarity to yeast SAM37 and has been implicated in mitochon-
drial protein import (Armstrong et al., 1997; Abdul et al., 2000).
METAXIN1 and METAXIN2 in mammals have been reported to
be involved in the import of b-barrel proteins but in a different
complex compared with SAM50 (Kozjak-Pavlovic et al., 2007).
METAXIN protein has been shown to be present in Arabidopsis
mitochondria (Lister et al., 2004), but no role in mitochondrial
protein import or sorting has been demonstrated.
Another notable difference between yeast and higher eukary-
otes, such as plants and mammals, is that the yeast subunits of
the TOM, TIM, and SAM complexes are encoded by single
nuclear genes, while in higher organisms, components of TOM,
TIM, and SAM are often encoded by small multigene families. It is
not clear what the function of these multigene families are,
whether they represent functionally distinct isoforms or provide
greater ability for regulation of these components at a transcrip-
tional level (i.e., subfunctionalization verses neofunctionaliza-
tion). In Drosophila melanogaster, it has been documented that
TOM20 and TOM40 are each encoded by two differentially
expressed genes (Hwa et al., 2004). Detailed expression analysis
of almost all genes encoding mitochondrial protein import com-
ponents in Arabidopsis indicates differential expression patterns
of genes within each family (Lister et al., 2004). Furthermore,
functional analysis of the Arabidopsis TIM17 gene family sug-
gests some differences in function based on the ability to
complement a tim17 mutant in yeast (Murcha et al., 2003).
Although yeast is an excellent model to provide the basic
mechanism of how proteins are imported into mitochondria, it
cannot give insights into the functions of nonhomologous com-
ponents in other organisms, such as plants, in which the putative
receptor components are not orthologous to yeast receptor
subunits. Furthermore, the yeast model system cannot be used
to explore any functional diversification of these multiple genes
encoding import components. Here, we investigate the function
of three outer mitochondrial membrane proteins in Arabidopsis
with respect to protein import into mitochondria. We demon-
strate that the three highly expressed TOM20 isoforms in
Arabidopsis are predominantly functionally equivalent but dis-
play limited specialization and together are important, but not
essential, for the import of a wide range of mitochondrial pro-
teins. Furthermore, we demonstrate that mtOM64 is a mitochon-
drial protein that can interact with a variety of precursor proteins,
increases in abundance when two or more TOM20 isoforms are
inactivated, and plays a role in the import for at least some
mitochondrial proteins. Finally, we identify that METAXIN inter-
acts with a wide variety of precursor proteins and is involved in
their import, and results suggest that it plays more than one role
in the import and assembly of proteins into mitochondria.
RESULTS
The TOM20 Gene Family Encodes Functionally Redundant
Proteins Involved in Mitochondrial Protein Import
TOM20 is encoded by four paralogous genes in the Arabidopsis
genome, TOM20-1 to TOM20-4 (Werhahn et al., 2001). TOM20-1
and TOM20-3 are tandemly duplicated genes, the predicted
proteins of which display 60% amino acid identity. TOM20-2,
TOM20-3, and TOM20-4 are highly expressed in diverse plant
organs (Lister et al., 2004), whereas by contrast, TOM20-1 tran-
script is rarely detectable (see Supplemental Figure 1A online)
(Lister et al., 2004; Murcha et al., 2007), and TOM20-1 is the only
TOM20 protein that has not been directly identified in isolated
plant mitochondria (Werhahn et al., 2001; Heazlewood et al.,
2004; Lister et al., 2004). Antibodies raised against TOM20-3
cross-react with in vitro–translated TOM20-1 (see Supplemental
Figure 1B online), but TOM20-1 could not be detected with this
antibody in mitochondria isolated from whole seedlings of any of
the Arabidopsis genotypes used in this study. Mitochondria were
isolated from wild-type Arabidopsis (ecotype Columbia-0) and
two independent T-DNA insertion lines of each highly expressed
TOM20 gene (Sessions et al., 2002; Alonso et al., 2003; Rosso
et al., 2003), and the absence of transcript derived from the
specific TOM20 isoforms was verified by RT-PCR (data not
shown) and immunodetection of total mitochondrial protein sam-
ples with specific antibodies raised to each of these TOM20
isoforms (Figure 1A). No severe phenotypic abnormalities were
observed in any of the single insertion mutants; however, tom20-2
had a slightly delayed flowering time, 4 to 7 d later than wild-type
plants (Figure 2A).
3740 The Plant Cell
In vitro import of radiolabeled plant mitochondrial precursor
proteins into mitochondria isolated from wild-type and tom20
plants was performed. While the selection of precursor proteins
represented a wide range of mitochondrial protein import path-
ways (general import pathway, alternative oxidase [AOX] and the
10-kD protein of the small mitochondrial ribosomal subunit
[RPS10]; carrier import pathway, mitochondrial phosphate car-
rier [PiC]; dual-targeted proteins, glutathione reductase [GR];
plant specific protein, FAd-subunit of mitochondrial ATP syn-
thase) (Murcha et al., 1999; Heazlewood et al., 2003), no signif-
icant differences in the rate of protein import were observed
between wild-type and tom20 mitochondria for any precursor
protein (see Supplemental Figure 2 online). Therefore, none of
the Tom20 proteins alone appeared to have an essential function
in mitochondrial protein import.
Different TOM20 Isoforms Display Some Precursor
Recognition Specificity
The single TOM20 isoform mutant plants were crossed to gen-
erate double TOM20 knockout plants deficient in each combi-
nation of the three highly expressed TOM20 isoforms. Notably,
the resulting double knockout plants were viable and did not
display severe phenotypic abnormalities, although again slight
delays in flowering time were noticeable (Figure 2A). Mitochon-
dria were isolated from wild-type and double tom20 knockout
plants and the protein complexes solubilized by digitonin treat-
ment and separated by one-dimensional blue native PAGE
(BN-PAGE), followed by immunodetection of the remaining
TOM20 protein (Figure 1B). TOM20-3 in both wild-type mitochon-
dria and the tom20-2 tom20-4 mitochondria was detected in a
protein complex of a similar apparent molecular mass, indicating
that the loss of two TOM20 isoforms did not disrupt the integrity or
structure of the TOM complex. This was also observed for
TOM20-4 in wild-type and tom20-2 tom20-3 mitochondria. Re-
sidual Coomassie blue staining of the mitochondrial inner mem-
brane respiratory complexes I, III, IV, and V enabled estimation of
the TOM complex molecular mass at ;230 kD, based on a
comparison with a previous study of potato (Solanum tuberosum)
mitochondrial membrane protein complexes (Jansch et al., 1998).
Interestingly, a second complex containing TOM20-2 was always
detected in wild-type mitochondria (Figure 1B, open arrowhead),
indicating that some TOM20-2 is located in a higher molecular
weight complex. Furthermore, in mitochondria isolated from
tom20-3 tom20-4 plants, a larger proportion of TOM20-2 was
present at a higher molecular weight, abolishing the two discrete
complexes and forming a continuous distribution of larger com-
plexes. Thus, when mitochondria only contain TOM20-2 it ap-
pears to lead to an ectopic size distribution of TOM complexes.
The rate of protein import into mitochondria with only one
functional TOM20 protein was measured in vitro for a range of
plant mitochondrial precursor proteins (Figure 2B). The import of
TOM40 and PiC was unaffected; however, for the other precur-
sor proteins, significant differences in the rate of protein import in
various mutants were observed (P < 0.05). AOX import into
tom20-2 tom20-3 and tom20-2 tom20-4 mitochondria was re-
duced to 71 and 64%, respectively, compared with AOX import
into wild-type mitochondria. FAd was imported into tom20-2
tom20-4 mitochondria at 85% of the wild-type rate of import.
Finally, the import of the dual-targeted protein GR into tom20-2
tom20-3 mitochondria was 31% higher after 10 min and 71%
higher after 20 min, at which the rate was still linear. Therefore,
generation of mutant plants with only one functional TOM20
protein revealed that a functional TOM complex can be formed
with a single TOM20 and that different TOM20 isoforms display
some precursor recognition specificity that is evident when
import of a variety of precursor proteins is assessed.
Tom20 Is a Nonessential Protein
Plants were generated that lacked all the three highly expressed
TOM20 isoforms, tom20-2 tom20-3 tom20-4 (tom20 triple knockout).
Figure 1. The TOM Complex Can Form with Only One TOM20 Isoform.
(A) Immunodetection of Tom20 isoforms in mitochondrial protein sam-
ples isolated from wild-type and tom20 T-DNA insertional mutant plants
and separated by SDS-PAGE.
(B) Immunodetection of Tom20 proteins in mitochondrial protein sam-
ples isolated from wild-type and tom20 double knockout plants after
digitonin solubilization and separation of protein complexes by first
dimension BN-PAGE. The open arrowhead indicates the higher molec-
ular weight complex containing TOM20-2. The position and approximate
molecular mass of the inner membrane respiratory complexes I, III, IV,
and V are indicated, as detected by the residual Coomassie blue staining
and reported in a previous study of potato mitochondrial protein
complexes (Jansch et al., 1998).
Mitochondrial Preprotein Import 3741
Figure 2. Arabidopsis TOM20 Is Encoded by a Multiple Gene Family of Predominantly Functionally Equivalent Isoforms That Plays a Role in the Import
of Preproteins into Mitochondria.
3742 The Plant Cell
These plants were viable but displayed a slightly slower growth
rate (Figure 2A). In vitro import of radiolabeled precursor proteins
into mitochondria isolated from tom20 triple knockout plants
indicated that TOM20 is involved in the import of a diverse range
of mitochondrial precursor proteins (Figure 2C). Import of AOX,
TOM40, and PiC was reduced to only 20 to 30% of the amount of
import into wild-type mitochondria (P < 0.01), indicating that
TOM20 is involved in the general, carrier, and outer membrane
b-barrel protein import pathways. However, the import of GR
was unaffected, and import of FAd did not decrease significantly
(P > 0.05), indicating that TOM20 has little or no involvement in
the import of these precursor proteins or that its absence can be
compensated for completely by another import component.
mtOM64 Is Involved in Mitochondrial Protein Import
mtOM64 is a protein anchored in the mitochondrial outer mem-
brane that has 67% protein sequence identity to At TOC64-III, a
chloroplast outer envelope protein import receptor (Chew et al.,
2004; Qbadou et al., 2006). Sequence analysis indicated that
mtOM64 has three C-terminal tetratricopeptide repeat (TPR)
motifs, resembling the C-terminal TPR domains of At TOC64-III
and TOM70 that function to recognize chloroplast and mito-
chondrial proteins bound to HSP90 (Young et al., 2003; Qbadou
et al., 2006). To investigate the role of mtOM64, two Arabidopsis
lines with independent T-DNA insertions in the coding region of
mtOM64 were obtained and a specific antibody was raised to
verify the absence of mtOM64 protein in the knockout plants
(Figure 3A). BN-PAGE analysis revealed that mtOM64 did not
migrate with a complex, but rather it was consistently detected at
the bottom of the gel, indicating that it did not form part of a larger
complex under the conditions tested (data not shown). In vitro
protein import reactions were conducted with a wide range of
mitochondrial precursor proteins into mitochondria isolated from
the two mtom64 plant lines (Figure 3B). In comparison with wild-
type mitochondria, no difference in the rate of protein import was
observed for AOX, PiC, GR, and TOM40. However, the import of
FAd was consistently 30 to 40% lower in plants lacking mtOM64
(P < 0.05), indicating that it is involved in the import of this plant-
specific protein. This is in agreement with a previous study that
suggested that FAd does not solely rely on TOM20 for import
(Dessi et al., 1996; Murcha et al., 1999).
To investigate the interaction of mtOM64 with mitochondrial
precursor proteins, import competition experiments were per-
formed with mtOM64 competitor protein that was synthesized in
an in vitro wheat germ lysate transcription/translation system.
First, radiolabeled precursor proteins were preincubated with
expressed competitor proteins in a wheat germ lysate mix (wheat
germ lysate resuspended in wheat germ reconstitution buffer),
either mtOM64 or an equivalent volume, and a quantity of
b-glucuronidase (GUS). Following preincubation, the precursor/
competitor protein mix was added to wild-type mitochondria
under conditions that support import, and the amount of pre-
cursor protein imported after 10 min was quantitated (Figure 3C).
Preincubation with the wheat germ reconstitution buffer alone
decreased protein import of AOX and FAd by 69 and 53%,
respectively (see Supplemental Figure 3 online). Preincubation
with wheat germ lysate mix inhibited AOX import by 65%,
indicating that the wheat germ reconstitution buffer, and not the
wheat germ lysate, was responsible for the decrease in AOX im-
port. By contrast, preincubation with the wheat germ lysate mix
did not inhibit FAd import, indicating that a factor present in the
wheat germ lysate stimulates FAd import, thus abrogating the in-
hibitory effect of the wheat germ reconstitution buffer. To control
for the effects of wheat germ lysate, wheat germ reconstitution
buffer, and protein overexpression, an identical volume of wheat
germ lysate mix in which GUS was overexpressed was used as a
control competitor protein; thus, the specific effect of mtOM64
on the import of a variety of proteins could be assessed. Prein-
cubation of AOX or TIM23 with the mtOM64 competitor protein
did not result in a significant change in the amount of precursor
protein import relative to the GUS control. However, preincuba-
tion of FAd with mtOM64 resulted in a significant reduction (P <
5.0 3 10�4) of FAd import into mitochondria of 32% compared
with GUS (Figure 3C), in close agreement with the decrease in the
amount of import into mtom64 mitochondria (Figure 3B). There-
fore, exogenous mtOM64 protein was able to specifically com-
pete with the FAd precursor protein but not other precursors
tested. Preincubation of FAd with the chloroplast import receptor
At TOC64-III did not affect FAd import, in contrast with the 32%
reduction after preincubation with mtOM64 (Figure 3D), again
indicating a specific effect of mtOM64. The ability of mtOM64 to
compete with the FAd precursor could result either from a direct
interaction with this precursor or interaction with a common compo-
nent on the outer membrane that enables import into mitochondria.
METAXIN Is an Outer Mitochondrial Membrane Protein
Essential for Normal Cellular Development, Starch
Metabolism, and Plant Growth
Although the tom20 triple and mtom64 knockout plants ap-
peared to interact with a range of precursor proteins, the lack of a
Figure 2. (continued).
(A) Five-week-old Arabidopsis plants with different genotypes indicated. Plants lacking Tom20-2 display a delayed-growth phenotype.
(B) [35S]-labeled precursor proteins AOX, FAd, TOM40, GR, and PiC were incubated with mitochondria isolated from wild-type and mutant plants
deficient in two of the TOM20 isoforms under conditions that support import. Aliquots were removed at 2, 5, 10, and 20 min and treated with PK. PK-
protected mature radiolabeled protein was quantitated at each time point and normalized to the highest time point for replicate experiments (n $ 3 6
SE). Where indicated, mutant mitochondria had a significantly lower amount of protein import than wild-type mitochondria, with a P value < 0.05 (*) and
0.01 (#) using Student’s t test. m, mature protein; p, precursor protein.
(C) [35S]-labeled precursor proteins AOX, FAd, TOM40, GR,x and PiC were incubated with mitochondria isolated from wild-type and tom20-2 tom20-3
tom20-4 plants (triple KO). The import time course and analysis were performed as above. Where indicated, mutant mitochondria had a significantly
lower amount of protein import than wild-type mitochondria, with a P value < 0.01 (#) and 0.005 (circles) using Student’s t test.
Mitochondrial Preprotein Import 3743
striking phenotype, as observed previously when plastid import
receptors were inactivated (Soll and Schleiff, 2004; Bedard and
Jarvis, 2005; Kessler and Schnell, 2006), suggested that addi-
tional components may also be present. Experiments were
conducted to identify additional mitochondrial outer membrane
proteins involved in protein import. As human METAXIN1 has
previously been implicated in mitochondrial protein import
(Armstrong et al., 1997), the role of Arabidopsis METAXIN was
investigated. Arabidopsis lines with independent T-DNA inser-
tions in the METAXIN coding sequence were obtained and the
Figure 3. mtOM64 Is a Mitochondrial Preprotein Import Component That Is Involved in the Import of FAd into Mitochondria.
(A) Immunodetection of mtOM64 in mitochondrial protein samples isolated from wild-type and mtom64 T-DNA insertional mutant plants and separated
by SDS-PAGE.
(B) [35S]-labeled precursor proteins AOX, FAd, Tom40, GR, and PiC were incubated with mitochondria isolated from wild-type and mtom64 plants under
conditions that support protein import. Aliquots were removed at 2, 5, 10, and 20 min and treated with PK. PK-protected mature radiolabeled protein was
quantitated at each time point and normalized to the highest time point for replicate experiments (n $ 3 6 SE). Where indicated, mutant mitochondria had a
significantly lower amount of protein import than wild-type mitochondria, with a P value < 0.05 (*), 0.02 (#), and 0.01 (circles) using Student’s t test.
(C) [35S]-labeled precursor proteins AOX, FAd, and TIM23 were preincubated with overexpressed GUS or mtOM64 competitor protein prior to
incubation with mitochondria from wild-type plants. PK-protected mature radiolabeled protein was quantitated and normalized against the amount of
protein imported after preincubation with GUS (n ¼ 3 6 SE). Where indicated, the mtOM64 competitor protein decreased protein import significantly
compared with GUS, with a P value < 5.0 3 10�4 (*) using Student’s t test.
(D) [35S]-labeled FAd precursor protein was preincubated with overexpressed GUS, At TOC64-III, or mtOM64 competitor proteins prior to incubation
with mitochondria from wild-type plants. PK-protected mature radiolabeled FAd was quantitated and normalized against the amount of protein imported
after preincubation with GUS. Lane 1, AOX precursor only; lane 2, AOX precursor incubated with GUS; lane 3, AOX precursor incubated with At
TOC64-III; lane 4, AOX precursor incubated with mtOM64. Where indicated, the competitor protein decreased protein import significantly compared
with GUS, with a P value < 0.01 (*).
3744 The Plant Cell
absence of METAXIN transcript confirmed (see Supplemental
Figure 4 online). metaxin plants displayed severe phenotypic
abnormalities following leaf emergence, including diminished
growth, abnormal leaf morphology and ectopic floral develop-
ment and sterility (Figures 4A and 4B). Iodine staining of plants at
the end of both the light and dark photoperiods revealed that
metaxin plants accumulated higher levels of starch (Figure 4C).
Light microscopy of leaf cross sections indicated that the me-
sophyll cells of the metaxin plants contained more chloroplasts
compared with the wild type (Figures 4D and 4E). Transmission
electron microscopy of leaf mesophyll cells revealed large starch
deposits within the chloroplasts of metaxin cells, which was not
observed in wild-type cells (Figures 4F and 4G). Genetic trans-
formation of the METAXIN cDNA sequence under the 35S
promoter of Cauliflower mosaic virus into metaxin plants rescued
the mutant phenotype (data not shown). Sequence analysis
indicated that METAXIN has two highly hydrophobic segments
near the C terminus, possibly acting as transmembrane regions
that anchor the protein in a membrane (Figure 4H). Arabidopsis
suspension cell culture was biolistically transformed with a
chimeric construct that fused green fluorescent protein (GFP)
to the N terminus of METAXIN. The full-length METAXIN protein
was able to direct N-terminal GFP to mitochondria, as indicated
by the colocalization of GFP with the mitochondrial-targeted
AOX-RFP (red fluorescent protein) control construct (Figure 4H).
Interestingly, the GFP signal formed hollow circular structures,
suggesting that the GFP was targeted only to the outer mito-
chondrial membrane. These structures closely resemble those
observed by Setoguchi et al. (2006) upon immunofluorescence
microscopy–based detection of the mammalian mitochondrial
outer membrane proteins TOM22 and Bak.
To independently confirm the outer membrane localization of
METAXIN, intact wild-type mitochondria were isolated and incu-
bated in increasing concentrations of proteinase K (PK). Immu-
nodetection of TOM20-2 and TOM20-4 using specific polyclonal
antibodies raised to their cytosolic domains revealed that
1.7 mg/mL PK resulted in the complete digestion of the cyto-
solic portion of these proteins (Figure 4I). Immunodetection of
METAXIN by a specific polyclonal antibody raised to the pre-
dicted METAXIN cytosolic domain indicated that the cytosolic
portion of METAXIN was completely degraded in the presence of
53.3 mg/mL of PK (Figure 4I). At up to 106.7 mg/mL PK, there was
no noticeable degradation of VDAC (voltage-dependent anion
channel) or AOX, which are located in the outer membrane and on
the matrix side of the inner mitochondrial membrane, respec-
tively, indicating that the PK did not compromise the integrity of
either of the mitochondrial membranes at the concentrations
used. Therefore, METAXIN is accessible to externally added PK,
indicating that the predicted cytosolic domain to which the
antibody was raised is exposed to the cytosol. Membrane pro-
tein complexes from both wild-type and metaxin mitochondria
were solubilized by digitonin and separated by one-dimensional
BN-PAGE, followed by immunodetection of METAXIN, TOM20,
and the COXII subunit (cytochrome c oxidase) of mitochondrial
respiratory Complex IV (Figure 4J). COXII colocalized with Com-
plex IV, as determined by Coomassie blue staining of the res-
piratory complexes (data not shown), whereas METAXIN was
detected in a high molecular weight complex that is distinct from
the smaller TOM complex (Figure 1B). Furthermore, this high mo-
lecular weight complex could not be detected in metaxin mito-
chondria, indicating that it is an authentic mitochondrial protein
complex containing METAXIN. Therefore, while METAXIN is
exposed to the cytosol on the outer mitochondrial membrane,
it is located in a distinct complex that has not previously been
characterized in plant mitochondria.
Mitochondria from Metaxin Plants Have Reduced Rates
of Protein Import
A range of radiolabeled mitochondrial proteins were incubated
with mitochondria isolated from wild-type and metaxin plants
(Figure 5). As the yeast SAM37 forms part of the SAM complex,
the import of two b-barrel proteins, TOM40 and VDAC, was also
studied. The rate of protein import into metaxin mitochondria was
dramatically reduced for all precursors tested, especially TOM40
and VDAC. This indicates that METAXIN is involved in the import
of these proteins into mitochondria, either because it is directly
required for import by all of these mitochondrial proteins or
because it is required for correct TOM40 import and assembly
and thus needed for generation of a functional general import
pore, as is observed for SAM37 in yeast (Paschen et al., 2003;
Wiedemann et al., 2003). Although the decrease in protein import
of all the precursor proteins may be due to insufficient import or
assembly of TOM40, two-dimenstional PAGE analysis indicated
that TOM40 was present in the same amount in metaxin plants as
in the wild type (see Supplemental Figure 6 online). This indicates
that the inactivation of METAXIN does not completely block the
import of TOM40.
As it cannot be ruled out that the decrease in the rate of protein
import into metaxin mitochondria was a downstream conse-
quence of the disruption of TOM complex structure/organization
or a process in mitochondria not directly related to protein import,
an alternative approach was required to determine if it played any
direct role in the import of a variety of precursor proteins destined
to various intramitochondrial locations. METAXIN was expressed
in a wheat germ lysate and preincubated with radiolabeled mito-
chondrial precursor proteins before incubation with mitochondria
to determine if exogenous METAXIN could compete with mito-
chondria for interaction with the precursor proteins. The addition
of METAXIN resulted in a large decrease in the import of diverse
precursor proteins into mitochondria, relative to the amount of
protein import when precursors were incubated with GUS (Figure
6A). METAXIN preincubation inhibited the import of the general
pathway proteins AOX and RPS10, the carrier pathway protein
PiC, and the dual-targeted GR by 50 to 70%, while the b-barrel
proteins TOM40 and VDAC were imported 30 to 40% less.
Notably, very little reduction (;10%) was observed for FAd, and
TIM23 import was not significantly reduced (P > 0.1), indicating
that the addition of METAXIN to the in vitro protein import reac-
tion was not causing general disruption of the protein import
machinery or general import pore but was likely specifically inter-
acting with certain precursor proteins.
Comparative sequence alignment of plant METAXIN, animal
METAXIN1, and fungal SAM37 protein sequences from nine
diverse species revealed that the sequence of SAM37 from the
Mitochondrial Preprotein Import 3745
Figure 4. METAXIN Is an Outer Mitochondrial Membrane Protein Required for Normal Plant Metabolism, Growth, and Development.
(A) METAXIN-deficient Arabidopsis (metaxin) displays retardation of growth.
(B) metaxin is sterile and displays floral abnormalities, including incomplete anther and style maturation.
(C) Iodine staining of starch at 0 and 12 h after initiation of the light photoperiod.
(D) Light microscopy of wild-type leaf cross section (arrow indicates chloroplast).
(E) Light microscopy of metaxin leaf cross section (arrows indicate chloroplasts).
(F) Transmission electron microscopy of wild-type leaf mesophyll cell.
(G) Transmission electron microscopy of metaxin leaf mesophyll cell (arrows indicate starch granules within chloroplast).
(H) Subcellular localization of METAXIN was tested by fluorescence microscopy visualization of Arabidopsis suspension cells biolistically
cotransformed with plasmids encoding NAOX-RFPC and NGFP-METAXINC. TM, transmembrane domain.
3746 The Plant Cell
fungi Schizosaccharomyces pombe and Aspergillus clavulata
were more similar to the animal METAXIN1 proteins than to yeast
SAM37 (Figure 6B; see Supplemental Figure 5 online). Two puta-
tive conserved domains were identified at similar positions in
each protein, in plant METAXIN the N-terminal GST-N-METAXIN,
and C-terminal GST-C-METAXIN domains, and in animals/fungi
the N-terminal GST-N-METAXIN1-like and C-terminal GST-C-
METAXIN1-3 domains (Figure 6B) (Marchler-Bauer and Bryant,
2004). Alignment of the METAXIN/SAM37 protein sequences
revealed regions of significant sequence similarity in these
domains. Furthermore, a region of 40 to 52 amino acids from
the C-terminal end of the GST-N-METAXIN domain displayed
significant similarity between all species, suggesting it may be
important for protein function (see Supplemental Figure 5 online).
Progressive truncations of METAXIN from the C terminus were
made to disrupt the three putative conserved domains, and the
proteins were translated in a wheat germ lysate (Figure 6C).
These METAXIN deletions were used in competition import
assays with wild-type mitochondria to ascertain which regions
of the METAXIN protein were required for it to compete with
mitochondrial-located METAXIN for the import of radiolabeled
AOX (Figure 6C). The first 112 amino acids of METAXIN inhibited
AOX import; however, further deletion to only the first 72 or
37 residues of METAXIN abolished the competitive inhibition of
AOX import, suggesting that the conserved region (or competitor
domain) identified between amino acids 72 and 112 may be
important for interaction of the METAXIN protein with AOX
(Figure 6C). Preincubation with METAXIN that lacked only
the competitor domain resulted in a significant decrease in
import compared with GUS, but the amount of import inhibition
was less than when full-length METAXIN was used as a com-
petitor protein, further supporting a role for this region in the
import of preproteins across the outer membrane. Finally, in-
creasing the concentration of the METAXIN1-112 competitor
protein resulted in increased levels of AOX import inhibition
(Figure 6D).
Figure 4. (continued).
(I) Mitochondria were purified from wild-type plants and incubated with increasing concentrations of PK. The mitochondrial proteins were separated by
SDS-PAGE and probed with specific antibodies as indicated.
(J) Immunodetection of CoxII and METAXIN in mitochondrial protein samples isolated from wild-type and metaxin plants after digitonin solubilization
and separation of protein complexes by first dimension BN-PAGE. The position and approximate molecular mass of the inner membrane respiratory
complexes I, III, IV, and V are indicated.
Figure 5. METAXIN-Deficient Mutant Plants Have Reduced Rates of Mitochondrial Protein Import.
[35S]-labeled precursor proteins TOM40, VDAC, AOX, FAd, PiC, and GR were incubated with mitochondria isolated from wild-type and METAXIN-
deficient mutants under conditions that support protein import. Aliquots were removed at 2, 5, 10, and 20 min and treated with PK. PK-protected mature
radiolabeled protein was quantitated at each time point and normalized to the highest time point for replicate experiments (n $ 3 6 SE). Where indicated,
mutant mitochondria had a significantly lower amount of protein import than wild-type mitochondria, with a P value < 0.05 (*), 0.01 (#), and 0.001 (circles)
using Student’s t test.
Mitochondrial Preprotein Import 3747
Figure 6. The Cytosolic Portion of METAXIN Can Interact with Mitochondrial Precursor Proteins.
(A) [35S]-labeled precursor proteins AOX, RPS10, TOM40, VDAC, PiC, TIM23, FAd, and GR were preincubated with overexpressed GUS or metaxin
competitor protein prior to incubation with mitochondria from wild-type plants under conditions that support protein import. PK-protected mature
radiolabeled protein was quantitated and normalized against the amount of protein imported after preincubation with GUS (n ¼ 3 6 SE). Where
indicated, the METAXIN competitor protein decreased protein import significantly compared with GUS, with a P value < 0.05 (*), 0.02 (#), 0.001 (circles)
using Student’s t test.
(B) Phylogenetic tree of protein sequences of plant METAXIN, animal METAXIN1, and fungal SAM37/METAXIN from Arabidopsis, M. truncatula,
O. sativa, C. elegans, A. mellifera, M. musculus, A. clavulata, S. pombe, and S. cerevisiae. METAXIN/SAM37 proteins from plants, animals, and fungi
display overlapping GST-N-METAXIN/GST-N-METAXIN1-like and GST-C-METAXIN/GST-C-METAXIN1-3 conserved domains.
(C) [35S]-labeled AOX precursor protein was preincubated with overexpressed GUS or truncated/deleted METAXIN proteins prior to incubation with
mitochondria from wild-type plants under conditions that support protein import. METAXIN (DComp) refers to a recombinant protein comprised of
the full-length METAXIN protein with the putative competitor domain between amino acids 72 and 112 removed. PK-protected mature radiolabeled
3748 The Plant Cell
TOM20, mtOM64, and METAXIN Interact with a Variety of
Precursor Proteins
To determine if these proteins could interact directly with precur-
sor proteins, pull-down and yeast two-hybrid interaction assays
were performed. For the pull-down assays, full-length TOM20-4,
METAXIN, and mtOM64 proteins were synthesized in a wheat
germ lysate and added to a variety of radiolabeled precursor
proteins. The interactions of each of these proteins with various
precursors was tested by the ability to pull down radiolabeled
precursor protein with antibodies raised against TOM20-4,
METAXIN, and mtOM64, respectively (Figure 7A). All three pro-
teins interacted with various precursor proteins, AOX, PiC, FAd,
TIM23, and TOM40. However, the precursor protein GR could
not be pulled down in this assay with any of the antibodies tested
(Figure 7A). Although it is not possible to compare how efficiently
each antibody pulls down each precursor due to variable anti-
body affinities, it was apparent that although the precursor pro-
tein FAd could be pulled down by all three antibodies, it was weak
in comparison to the other precursor proteins. For both AOX and
FAd, removal of the mitochondrial targeting signal resulted in
no interaction (Figure 7A). In contrast with the positive interaction
detected with the various precursor proteins and TOM20,
METAXIN, and mtOM64, using the same lysate programmed to
synthesize GUS, or using the lysate alone (Figure 7A) resulted in
no detectable interaction, indicating that the antibodies were not
directly binding to any of the precursor proteins (Figure 7A). The
binding of the Protein A Sepharose to the antibody was con-
firmed with protein gel blotting (Figure 7B). To further determine
if these proteins interacted with mitochondrial precursor pro-
teins, a yeast two-hybrid interaction screen was performed. The
cDNAs encoding the three TOM20 proteins, METAXIN, mtOM64,
and At TOC64-III, were each cloned to produce a recombinant
fusion protein with the GAL4 binding domain (bait), while cDNAs
encoding the precursor proteins were cloned to produce a fusion
protein with the GAL4 activation domain (prey). Interactions were
determined by the ability to grow without the addition of His to
the media and the ability to grow in the absence of adenine to
give a red/orange color. All mitochondrial precursor proteins
supported growth in the absence of His when TOM20, METAXIN,
or mtOM64 was used as bait, and colony numbers were typically
5- to 10-fold higher than that obtained when At TOC64-III was
used as bait (data no shown). To visualize the difference, colonies
were grown in the absence of adenine (Figure 7C). Whereas a
red/orange color is clearly evident for all mitochondrial precursor
proteins with the mitochondrial proteins as bait, At TOC64-III
colonies are clearly white. Notably, nonrecombinant bait and/or
prey constructs did not produce any interaction (see Supple-
mental Figure 7 online). Removing the mitochondrial targeting
signal from AOX or FAd resulted in no interaction. One exception
was when GR was used as the prey fusion protein; a weak orange
color was evident with At TOC64-III as bait. Arabidopsis GR is a
dual-targeted protein (Chew et al., 2003a), although the plastid
receptor for this protein has not yet been determined. It contains
the consensus phosphorylation motif in the targeting signal that
has been reported to increase targeting ability to chloroplasts
(Chew et al., 2003a, 2003b; Martin et al., 2006). Thus, it is possi-
ble that it can interact with At TOC64-III, although the color was
weak compared with the color development with mitochondrial
precursor proteins and mitochondrial outer membrane compo-
nents. In summary, the two assays show the ability of the three
mitochondrial proteins, TOM20, METAXIN, and mtOM64, to in-
teract with a variety of mitochondrial precursor proteins.
A Flexible Regulatory Response to Mitochondrial Protein
Import Dysfunction
Adaptive responses of the plant cell to ablation of the different
protein import components were monitored using antibodies
raised to each TOM20 isoform, mtOM64, METAXIN, and several
other mitochondrial proteins (Figure 8A). Overall, these results
indicated a complicated series of responses. The absence of any
one of the TOM20 isoforms did not result in an increase in the
abundance of either of the remaining two TOM20 proteins (Figure
8A). However, in plants lacking two TOM20 isoforms, the amount
of the remaining TOM20 protein increased at least twofold,
supporting the observation from in vitro protein import experi-
ments (Figure 2B) that the TOM20 isoforms are largely function-
ally redundant. Furthermore, the amount of METAXIN was
increased in each TOM20 double mutant. The level of the inner
mitochondrial membrane translocase component TIM17 in-
creased in tom20-2 tom20-4, tom20-3 tom20-4, and the tom20
triple knockout, indicating that the retrograde regulatory mech-
anism that increases TOM20 abundance may also control other
mitochondrial protein import components. Interestingly, mtOM64
abundance increased in all tom20 plants except tom20-2 (Figure
8A). Together, the alteration in abundance of each component is
consistent with the proposal of overlapping roles for these pro-
teins. Notably, no signal was evident in the tom20 triple knockout
when probed with the TOM20-3 antibody. As this antibody
clearly interacts with TOM20-1 (see Supplemental Figure 1B
online), and as the abundance of TOM20-2, TOM20-3, and
TOM20-4 increased in the respective TOM20 double knockout
lines, it appears that TOM20-1 abundance is not being increased
Figure 6. (continued).
AOX was quantitated and normalized against the amount of protein imported after preincubation with GUS. Where indicated, the METAXIN competitor
protein decreased protein import significantly compared with GUS, with a P value < 0.01 (#) using Student’s t test. The circle indicates that the full-length
METAXIN competitor protein inhibited AOX import significantly more than METAXIN (DComp), with a P value < 0.02 using Student’s t test.
(D) [35S]-labeled AOX precursor protein was preincubated with overexpressed GUS or METAXIN1-112 (the N-terminal 112 amino acids of METAXIN) prior
to incubation with mitochondria from wild-type plants under conditions that support protein import. PK-protected mature radiolabeled AOX was
quantitated and normalized against the amount of protein imported after preincubation with GUS. Where indicated, the METAXIN1-112 competitor protein
decreased protein import significantly compared with GUS, with a P value < 0.01 (#) using Student’s t test. Circles indicate that addition of METAXIN1-112
competitor protein inhibited AOX import significantly more than the next lower concentration of competitor, with a P value < 0.01 using Student’s t test.
Mitochondrial Preprotein Import 3749
Figure 7. Interaction of Precursor Proteins with TOM20, mtOM64, or METAXIN.
(A) The ability of TOM20, mtOM64, or METAXIN to pull down precursor protein in solution was tested for various precursor proteins. The lanes indicate
the antibody used to pull down the precursor proteins from a solution containing the corresponding protein synthesized in the RTS lysate. The left-hand
panel represents the reaction where the RTS lysate was programmed to synthesize the target protein. The right-hand panel represents the negative
control, where RTS lysate was not programmed to synthesize any protein, and thus the respective pull-down reactions contained no TOM20, METAXIN,
mtOM64, or GUS, respectively. AOXDp and FAdDp represent altered precursor proteins where the mitochondrial targeting presequence is not present.
(B) Protein gel blot analysis of the pull-down reactions to verify that the target protein was being specifically pulled down. Pull-down reactions were
performed and 10% of this reaction was analyzed by protein gel blotting. For each protein synthesized in the RTS reaction, the pull-down reaction was
performed and separated by SDS-PAGE, blotted, and probed with antibodies to TOM20, METAXIN, mtOM64, and anti-rabbit IgG. Lanes 1 to 4 in each
panel represent lysate programmed to synthesize TOM20, METAXIN, mtOM64, and GUS, respectively. A positive product was detected with the
TOM20 and METAXIN antibody (indicated by cirlces). As the mtOM64 protein has a similar molecular mass to that of the heavy IgG chains, a specific
product for mtOM64 could not be resolved; the rabbit IgG molecules are present as these were used to pull down the target proteins.
(C) Yeast two-hybrid interaction assays. The mitochondrial outer membrane proteins TOM20-2, TOM20-3, TOM20-4, mtOM64, and METAXIN and the
chloroplast outer envelope protein At TOC64-III were cloned into the bait vector and tested for interaction with the five precursor proteins cloned into the
prey vector. A red/orange color indicates an interaction. AOXDp and FAdDp represent these precursor proteins where the mitochondrial targeting
presequence had been removed.
3750 The Plant Cell
perceptibly to compensate for the loss of the three highly expressed
isoforms. As TOM20-1 transcript abundance is extremely low (see
Supplemental Figure 1C online) and detection of the TOM20-1
protein has not been reported, it appears that this isoform does not
play an important role in mitochondrial protein import.
AOX protein abundance, a marker for retrograde signaling in
plant mitochondria (Rhoads et al., 2006), increased in all mutants
lacking TOM20-2, most noticeably in tom20-2/tom20-4 and
the tom20 triple knockout. The in vitro protein import experi-
ments revealed that TOM20-2 plays a specialized role in AOX
Figure 8. Protein Levels in Mutant Plants Indicate Retrograde-Regulated Compensation by TOM20 Isoforms, Disruption of the TOM Complex in
metaxin, VDAC Accumulation in the Cytosol of metaxin Cells, and Upregulation of mtOM64 and METAXIN When Multiple TOM20 Isoforms Are Depleted.
(A) Mitochondria were purified from wild-type and mutant plants, and the mitochondrial proteins separated by SDS-PAGE and probed with specific
antibodies to import components and other mitochondrial proteins.
(B) Detection of marker proteins in mitochondrial and cytosolic protein fractions from wild-type and METAXIN-deficient plants. Lane 1, the wild type;
lane 2, metaxin-1; lane 3, metaxin-2.
(C) Abundance of TOM40, VDAC, UBC (ubiquitin conjugating enzyme), and ACT2 (Actin2) transcripts in metaxin-1 and metaxin-2 rosette leaves relative
to wild-type levels. Where indicated, the transcript abundance in metaxin was significantly higher than in the wild type, with a P value < 0.05 (*) using
Student’s t test. cFBPase, cytosolic fructose-1,6-bisphosphatase; E1a, E1 a-subunit of pyruvate dehydrogenase.
Mitochondrial Preprotein Import 3751
recognition and import (Figure 2B). AOX is a marker of stress-
induced mitochondrial retrograde pathways in plants, and its
upregulation in tom20-2 indicates that this isoform alone appears
to trigger this response (Rhoads et al., 2006). Similarly, the E1a
subunit of pyruvate dehydrogenase (E1a) was less abundant in
mitochondria isolated from tom20-2/tom20-3 and the tom20
triple knockout, indicating that TOM20-2 and TOM20-3 may both
have a higher affinity for this mitochondrial protein. The levels of
the inner membrane uncoupling protein (UCP), VDAC, cyto-
chrome c oxidase subunit II (COXII), and heat shock protein 60
(HSP60) were unchanged in the tom20 mutant plants, including
the tom20 triple knockout, demonstrating that despite lacking all
TOM20 receptors, the mitochondria are able to import a wide
range of proteins to a final abundance very similar to wild-type
levels. This again demonstrates that TOM20 is not an essential
protein import component and that the plant cell lacking TOM20
is able to generate a mitochondrial proteome quite similar to
wild-type mitochondria. This may be achieved by the operation
of the other protein import components mtOM64 and METAXIN.
Furthermore, it suggests that the upregulation of TOM20,
mtOM64, and TIM17 in the tom20 double and triple knockout
plants is not the consequence of an extensive alteration of a wide
range of mitochondrial proteins, but rather the outcome of a
precise retrograde regulatory pathway that specifically targets
the mitochondrial protein import apparatus.
The mtom64 mitochondria did not display significant changes
in the abundance of the proteins tested, except that the abun-
dance of METAXIN was consistently higher (Figure 8A). The
metaxin mitochondria showed dramatic decreases in the abun-
dance of several mitochondrial proteins, including an almost
total absence of all TOM20 isoforms and mtOM64 and a large
reduction in TIM17 and UCP abundance (Figure 8A). Interest-
ingly, AOX protein levels increased significantly in the metaxin
mitochondria, as did the alternative respiratory pathway activity
catalyzed by AOX (Ho et al., 2007), suggesting that this protein is
required at a greater abundance in the mitochondria in response
to aberrant mitochondrial behavior. This is in agreement with
the suggestion that elevated AOX abundance in mitochondria
lacking TOM20-2 is an adaptation to increased stress. By con-
trast, the level of VDAC, E1a, COXII, and HSP60 were unaltered
compared with wild-type mitochondria, suggesting that despite
a partially dysfunctional protein import apparatus in metaxin, the
plant cell can achieve normal levels of many mitochondrial pro-
teins. To determine if the abundance of VDAC throughout the
plant cell was unaltered by ablation of METAXIN, cytosolic pro-
teins were isolated from wild-type and metaxin leaves and the
amount of VDAC measured by immunodetection (Figure 8B).
metaxin plants had a much higher abundance of VDAC in the
cytosol, in contrast with the very low levels of VDAC in the wild-
type cytosolic fraction. Equivalent abundance of the cytosolic
fructose-1,6-bisphosphatase between wild-type and metaxin
cytosolic fractions demonstrated that not all cytosolic proteins
in the mutant had increased abundance. Cytosolic accumulation
of mitochondrial E1a was not observed, indicating that the cyto-
solic accumulation does not occur for all mitochondrial proteins
and that METAXIN may be particularly important for the recog-
nition and import of VDAC. Together with the increased VDAC
and TOM40 transcript abundance in metaxin plants (Figure 8C),
this suggests that the metaxin cells upregulate the expression
of this critical protein to attain normal levels in mitochondria,
potentially by saturating the residual import capability of the
metaxin mitochondria. To determine if TOM40 was present in the
metaxin and tom20 triple knockout lines, two-dimensional iso-
electric focusing (IEF)/SDS-PAGE analysis was performed with
purified mitochondrial proteins, and the amount of TOM40 was
determined. Overall, there appeared to be no significant changes
in TOM40 abundance in any of the mutant plant lines (see
Supplemental Figure 6 online). Likewise, for the FAd subunit, it
was evident that it was present at normal levels in the mtom64
plants (see Supplemental Figure 6 online). Finally, it was evident
that AOX was more abundant in tom20-2, tom20-2 tom20-4,
tom20-2 tom20-3 tom20-4, and metaxin lines (Figure 8A), even
though import was reduced from ;30 to 80%, respectively. Thus,
the steady state levels of proteins present in mitochondria isolated
from 4-week-old mutant plants was not affected by the absence
of the respective import components, even though a reduced rate
of protein import for these proteins was measured in vitro.
DISCUSSION
Investigation of the function of TOM20, mtOM64, and METAXIN
using a variety of approaches indicated that they all play a role in
the import of proteins into mitochondria. Insertional inactivation
is a powerful tool to determine function but cannot distinguish
between primary (direct) and secondary (indirect) effects. Here,
we observed that (1) altering any one of the three components
resulted in changes in abundance of at least one of the other two
that could possibly compensate for the function (Figure 8); and
(2) in the metaxin mutant, a reduced amount of other compo-
nents was observed (Figure 8). To overcome these limitations, a
variety of alternative approaches was undertaken. The potential
for all three components to directly interact with precursor
proteins was demonstrated (Figure 7), which combined with
the import and competition assays, strongly suggests a model of
overlapping ability to interact with precursor proteins on the outer
surface of mitochondria. Thus, although the insertional inactiva-
tion of mtOM64 only affected the import of the FAd precursor
(Figure 3), the interaction assays indicate that it can bind and
affect the import of a variety of precursor proteins (Figure 7).
However, as METAXIN abundance increased in the mtom64
mutants (Figure 8) and TOM20 is present, an in vitro assay where
excess precursor protein is added the absence of a nonlimiting
component may result in normal rates of import being observed
even if that component can interact with precursor proteins when
present. It was notable that in the mtom64 mutants, only the
import of the FAd precursor was affected (Figure 3), and the
competition assays indicated that METAXIN competitor protein
could only compete weakly for the import of this precursor
protein (Figure 6). Thus, the upregulated abundance of METAXIN
and the fact that the import of the FAd precursor is still high in the
presence of the triple tom20 knockout (Figure 2C) are consistent
with a role for mtOM64 in the import of this precursor protein.
However, TOM20 and METAXIN can also play a role, as they can
interact directly with this precursor protein (Figure 7). mtOM64
may play a role in the import of other precursor proteins, but due
to the apparent functional redundancy of the import machinery,
3752 The Plant Cell
this is not detected in knockout mutants, and other approaches
are required to elucidate its interaction with these precursor
proteins, such as direct interaction assays. In the case of GR,
only the yeast two-hybrid assay indicated an interaction. The in
vitro import assays in the triple tom20 and mtom64 mutants
revealed no effect on import. Addition of full-length METAXIN
could compete for import, but this may be due to competition for
a common import component. Alternatively, as GR is located in
the intermembrane space and the matrix (Chew et al., 2003a,
2003b), it may interact with METAXIN via the import route to the
intermembrane space or directly with GR via the intermembrane
space exposed domains of METAXIN. A recent study of METAXIN
from human cell lines using coimmunoprecipitation revealed that
it interacted with an inner membrane protein present in a larger
complex with several other proteins (Xie et al., 2007), while
another study in humans using BN-PAGE concluded that it was
not in a complex with SAM50 (Kozjak-Pavlovic et al., 2007). Thus,
the role of METAXIN may differ considerably between species
and/or it may play a variety of roles in various organisms.
The Tom20 Multiple Gene Family
Duplication of the TOM20 gene family has apparently resulted in
limited functional specialization, as evidenced by the formation
of a second and larger complex containing TOM20-2 and differ-
ences in the rate of import of some precursor proteins. As the rice
genome encodes only a single TOM20 isoform, this gene family
duplication and subfunctionalization has most likely occurred
subsequent to the divergence of monocotyledonous and dicot-
yledonous plants. Studies on duplicated genes suggest that
subfunctionalization is an important transition state to neofunc-
tionalization and acts to increase the preservation of duplicated
genes (Rastogi and Liberles, 2005). TOM20 was identified in
plants from direct protein analysis of the isolated TOM complex
that contained TOM40 (Jansch et al., 1998). Subsequent analysis
of TOM20 indicated that it is not orthologous to yeast or mam-
malian TOM20 (Perry et al., 2006), thus necessitating reevalua-
tion of its role in mitochondrial protein import. Based on the fact
that the majority of the protein is exposed on the cytosolic side of
the outer membrane (Figure 4), that the nuclear magnetic reso-
nance structure of TOM20-3 indicates a very similar prese-
quence binding fold to mammalian TOM20 (Likic et al., 2005;
Perry et al., 2006), that inactivation of all three TOM20 isoforms
leads to a substantial decrease in the rate of protein import
(Figure 2), that TOM20 directly interacts with mitochondrial
precursor proteins (Figure 7), and that overexpressed Arabidop-
sis TOM20-3 can compete for protein import into yeast mito-
chondria (Perry et al., 2006), we conclude that TOM20 likely
functions as a mitochondrial protein import receptor.
Although we could not detect TOM20-1 protein and transcript
was not detected in a variety of materials we have previously
examined (see Supplemental Figure 1 online) (Lister et al., 2004;
Murcha et al., 2007), it is possible that its expression at a protein
level is below the limits of detection or limited to specific cell
types. An examination of 2509 arrays in Genevestigator indicates
that it is called present in 347 (14%) (Zimmermann et al., 2004),
although this does not incorporate false discovery rate correction
that should be used (Nettleton, 2006). Analysis of these arrays
indicated that TOM20-1 transcript abundance was higher in
roots compared with other organs. Thus, we performed quanti-
tative RT-PCR and could detect some expression (see Supple-
mental Figure 1C online); however, the corresponding protein
could not be detected in mitochondria isolated from the same
triple tom20 mutant plants (water culture) used to carry out the
in vitro import assays (Figure 2) or from a variety of other mito-
chondrial preparations (cell culture, water culture or plant or root
material; data not shown). Thus, given the reduction we observe
in import with the tom20 triple knockout for several precursor
proteins, combined with the fact that we cannot detect the
protein, it is unlikely that TOM20-1 is highly expressed and could
compensate to function as the predominant import receptor.
Is mtOM64 a Receptor?
The absence of a plant homolog of TOM70 has frequently been
noted as one of the fundamental differences in the plant mito-
chondrial import apparatus (Lister et al., 2005; Chan et al., 2006).
In this study, mtOM64 could not be identified in the TOM
complex by immunodetection (data not shown), suggesting
that any attachment is peripheral and that it is dynamically
associated with the TOM complex, as observed for the interac-
tion of At TOC64-III with the TOC complex (Schleiff et al., 2003).
Insertional inactivation, in vitro competition experiments with
overexpressed mtOM64, antibody inhibition assays, and direct
interaction assays all suggest a role for mtOM64 in the import of
proteins into mitochondria. For the competition experiments,
mtOM64 (or METAXIN) was synthesized in a wheat germ tran-
scription/translation system where waste products are continu-
ously removed to achieve high levels of protein expression. It has
been reported that a different wheat germ lysate system than the
one used in this study can have an inhibitory effect on protein
import into mitochondria and plastids (Schleiff et al., 2002; Dessi
et al., 2003). However, the following should be noted: (1) the FAd
precursor is as efficiently imported into mitochondria from a
wheat germ lysate compared with a rabbit reticulocyte lysate
(Dessi et al., 1996; Tanudji et al., 2001), and (2) a study analyzing
the inhibitory effect of wheat germ on import indicated that it is
due to the folding status of the mature part of the protein (Dessi
et al., 2003). Other precursor proteins can also be imported into
mitochondria from a wheat germ lysate (Biswas and Getz, 2004).
Thus, it appears that some formulations of the wheat germ
translation lysate contain factors that inhibit import, and, notably,
it has been reported that other factors, such as mitochondrial
import stimulating factor, can relieve this inhibition (Hachiya
et al., 1993). The type of wheat germ translation system used
here is optimized to produce enzymatically active protein. Over-
all, although addition of this lysate mix alone reduces the rate of
protein import, it can be readily used, as it is a plant-based lysate,
and the inhibition observed in these studies was dependent on
the mRNA used to program the lysate and thus was not a general
effect of the lysate itself on import as was the topic of investi-
gation in previous studies. Furthermore, as evidenced by the
immunodetection (Figure 8A) and two-dimensional IEF/SDS-
PAGE (see Supplemental Figure 6 online) analyses, the steady
state level of mitochondrial proteins is not directly related to the
in vitro rate of import.
Mitochondrial Preprotein Import 3753
Several independent approaches used here all indicate a role
for mtOM64 in the import of at least some mitochondrial proteins;
however, its exact role cannot yet be concluded. Although it
can clearly interact directly with a variety of precursor proteins,
the binding chain hypothesis for mitochondrial import proposes
that many components contain such binding properties. For in-
stance, it has been recently shown that the pore-forming SAM50
(TOB55) from yeast contains such a domain (Rehling et al., 2001;
Habib et al., 2007). The localization of mtOM64 on the cytosolic
face of the outer membrane is evidenced by digestion with
externally added protease (Chew et al., 2004) and supports
an interaction with precursor proteins on the outer face of
mitochondria. At TOC64-III, TOM70, and mtOM64 have three
C-terminal TPR motifs that are predicted to form a superhelical
structure (Scheufler et al., 2000). Recently, the C-terminal TPR
motifs of Arabidopsis At TOC64-III were demonstrated to medi-
ate its recognition of chloroplast precursor proteins via interac-
tion with the chaperone HSP90 (Qbadou et al., 2006). In this
study, the competitive inhibition of FAd import by mtOM64 was
not abolished by addition of geldanamycin, a chemical that binds
to HSP90 and inhibits its chaperone activity (data not shown)
(Young et al., 2003). Thus, it can be concluded that mtOM64
does specifically interact with mitochondrial precursor proteins
and therefore may function as a receptor in the early stages of
precursor recognition and import. mtOM64 is a rate-limiting com-
ponent for the import of the plant-specific protein FAd (Figure 3),
although this may be an indirect affect due to the inactivation of
mtOM64 altering the level of as yet unknown component in-
volved or rate-limiting for the import of this precursor protein.
A Multifunctional METAXIN?
As the yeast SAM complex contains two of the three outer mem-
brane proteins known to be essential for cell viability, SAM50 and
SAM35 (Milenkovic et al., 2004), it was surprising to find that only
SAM50 is highly conserved in diverse eukaryotic organisms.
Animals possess METAXIN1 and METAXIN2, which display just
36 and 28% sequence similarity to yeast SAM37 and SAM35,
respectively (Mus musculus sequences). METAXIN2 was dem-
onstrated to interact with METAXIN1 and to be peripherally
associated with the mitochondrial outer membrane (Armstrong
et al., 1999), akin to yeast SAM35 (Waizenegger et al., 2004).
Taken together, the sequence similarity and protein–protein
interactions suggest that the animal METAXIN proteins are
orthologs of SAM35 and SAM37. By contrast, Arabidopsis
possesses only one METAXIN protein, which displays 21%
sequence similarity to mouse METAXIN1 and only 11% similarity
to yeast SAM35. Thus, it appears that the plant METAXIN is a
highly diverged form of yeast SAM37. The location of Metaxin on
the outer mitochondrial membrane and sensitivity to externally
added protease (Figure 4), the ability of various regions of the
METAXIN protein to inhibit import of some precursor proteins
(Figure 6), and the ability to interact with precursors directly
(Figure 7) together strongly suggest that METAXIN can interact
with precursor proteins on the outside of mitochondria. However,
given that METAXIN is not located in the TOM complex with
TOM20 (Figure 4), it would be premature to conclude that it plays
a primary role as a preprotein receptor.
The accumulation of VDAC in the cytosol of metaxin plants
(Figure 8) and the negligible rate of VDAC and TOM40 import into
metaxin mitochondria in vitro (Figure 5) suggest that METAXIN
also performs a role in b-barrel protein import. The competition
and interaction experiments indicated that plant METAXIN can
bind both VDAC and TOM40 (Figures 6 and 7), and the abun-
dance of transcripts encoding these proteins increased signifi-
cantly in metaxin plants (Figure 8). Therefore, plant METAXIN likely
functions in a plant SAM complex, which remains to be charac-
terized. The significant sequence similarity of plant and animal
METAXIN proteins and the conservation of the plant GST-N-
METAXIN and GST-C-METAXIN motifs and the functional
Competitor domain in animal METAXIN and yeast SAM37 sug-
gests that animal METAXIN and yeast SAM37 may have some
functional similarities to the plant METAXIN. Mutant yeast cells
lacking SAM37 displayed reduced import of several mitochon-
drial proteins, and anti-SAM37 antibodies were able to inhibit
protein import, leading to the initial characterization of SAM37 as
an import receptor that cooperated with TOM70 to recognize a
range of mitochondrial proteins (Gratzer et al., 1995). Subse-
quently it was reported that SAM37 was not involved in the initial
binding to mitochondria of the inner membrane metabolite
carrier protein AAC, and its depletion did not affect the general
or carrier import pathways (Ryan et al., 1999). However, if TOM20
and TOM70 were still present, their receptor capabilities may
have compensated for the absence of SAM37. Indeed, inactiva-
tion of SAM37 was synthetically lethal with the deletion of either
TOM20 or TOM70 (Gratzer et al., 1995). Thus, the role of the
cytosolic domain of METAXIN remains unclear in other orga-
nisms; however, its ability to interact with a variety of precursor
proteins suggests it may play another role in addition to the
assembly of b-barrel proteins and thus interact with proteins on
both the outside and inside of the outer membrane. Notably, At
TOC64-III in chloroplasts has been proposed to play a role in
precursor binding on the cytosolic and inter envelope space
(Qbadou et al., 2007).
A Flexible Import Apparatus with Overlapping Specificity
Overall, TOM20, mtOM64, and METAXIN possess cytosolic
domains that are located on the outer face of the mitochondria
and are required for normal rates or protein import. Overex-
pressed protein competes for import, and they can interact with a
variety of precursor proteins, all indicating that it is likely that
TOM20, mtOM64, and METAXIN directly interact with mitochon-
drial precursor proteins to function in the initial stages of the
import process. Given the NMR structure of TOM20 combined
with the results presented here, it is likely that it is a receptor for at
least some precursor proteins, while mtOM64 and METAXIN
may play similar roles.
These findings provide a unique insight into the complexity of
mitochondrialpreprotein importmachinery inamulticellulareukary-
ote. Analysis of genome sequences from a variety of organisms
suggests that plants do not have orthologs to TOM20 and TOM70
and that the receptor domain of TOM22 is lacking, compared
with yeast and mammalian systems (Macasev et al., 2004). Here,
we have defined three plant outer mitochondrial membrane pro-
teins that may fulfill the roles of preprotein receptors to produce a
3754 The Plant Cell
flexible and redundant set of receptor subunits in plants. SAM37/
35 and METAXIN appear to play a role in b-barrel assembly in all
lineages but may have acquired lineage-specific functions as a
receptor subunit, especially evident in plants. Finally, the use of
very similar protein import components in both plant mitochon-
dria and plastids suggests coevolution of the import machineries
of both organelles, which may have been a significant impetus in
the development of this unique import apparatus.
METHODS
Plant Growth
All plants were grown at 228C under long-day conditions (16 h of 100 mE
m�2 s�1 light, 8 h dark), except for metaxin plants that were iodine stained
for starch content analysis, which were grown with a 12-h-light/12-h-dark
photoperiod. For T-DNA insertion line genotyping, Arabidopsis thaliana
seeds were grown on soil after stratification for 2 d. For mitochondrial
isolation, Arabidopsis seeds were sterilized in 70% (v/v) ethanol and 5%
(v/v) bleach/0.1% (v/v) Tween 20 and grown for 14 d on an orbital shaker
at 80 rpm in 80 mL of sterile liquid growth media (0.53 Murashige and
Skoog media, 0.53 Gamborgs B5 vitamins, 2% [w/v] sucrose, 50 mg/mL
cefotaxime, and 2 mM MES KOH, pH 5.7). As metaxin homozygotes are
infertile, seeds from plants heterozygous for the metaxin-1 or metaxin-2
null allele were sterilized as above and grown for 14 d on agar growth media
(13 Gamborgs B5 salts, 3% [w/v] sucrose, 50 mg/mL cefotaxime, 2 mM
MES KOH, pH 5.7, and 0.75% [w/v] agar). metaxin homozygote plants were
then transplanted to soil and grown until flowering. Columbia-0 control
plants were grown under the same conditions as metaxin.
Iodine Staining
For iodine staining of starch, plant tissue was boiled for 5 min in 80% (w/v)
ethanol, washed in water, and then incubated for 5 min in 50% (v/v)
Lugols’s solution (Sigma-Aldrich). Plant tissue was then destained for
90 min in water.
T-DNA Insertion Lines
The following T-DNA insertion lines were obtained from SALK (Alonso
et al., 2003), SAIL (Sessions et al., 2002), and GABI-KAT (Rosso et al.,
2003) collections and genotyped to confirm homozygosity for the T-DNA
insert: tom20-2 (At1g27390): SALK_067986, SALK_134973; tom20-3
(At3g27080): GABI_554C03, SAIL_88_A03; tom20-4: SALK_147093,
SALK_004057; mtOM64 (At5g09420): SALK_068772, SALK_089921;
and metaxin (At2g19080): SALK_107629, SALK_039892.
Mitochondrial Isolation
Approximately 10 g (fresh weight) of aerial tissue from soil-grown plants or
20 g (fresh weight) of 14-d-old seedlings from liquid-grown (water culture)
plants were used to isolate mitochondria as described previously (Day
et al., 1985), with 10 mM L-Cys added to the grinding medium. Typically,
10 g of soil-grown and 20 g of liquid-grown plant tissue yielded ;2 mg of
mitochondrial protein. For isolation of mitochondria for protein gel blot
analysis, BSA was omitted from the final washes. All mitochondria were
isolated from liquid-grown seedlings except for the metaxin genotypes,
for which aerial tissue was used due to sterility of the metaxin null mutant.
Cytosol Isolation
Rosette tissue was homogenized in grinding buffer without BSA (Day
et al., 1985), centrifuged at 2500g for 5 min, and then the supernatant
centrifuged at 40,000g for 40 min. Crude cytosolic supernatant was
centrifuged at 100,000g for 1 h at 48C. The supernatant was concentrated
in a >5 kD centrifugal filter unit (Millipore).
Immunodetection of Proteins, Pull-Down Assays, and Antibody
Inhibition of Import
Mitochondrial or cytosolic proteins (50 mg) were resolved by SDS-PAGE,
transferred to Hybond-C extra nitrocellulose membrane, and immuno-
detection performed as previously outlined (Murcha et al., 2005). Poly-
clonal antibodies were raised in rabbits against recombinant protein
encoded by the predicted cytosolic regions of METAXIN, mtOM64,
TOM20-2, and TOM20-4. TOM20-3 polyclonal antibody was obtained
from Trevor Lithgow (University of Melbourne, Vic, Australia) (Taylor et al.,
2003). Antibodies to HSP60 were obtained from Stress-Gen, and anti-
bodies to COXII and cFBPase were obtained from Agrisera. Monoclonal
antibodies against VDAC (PM035) and E1a (PM030) were obtained from
Tom Elthon (University of Nebraska, Lincoln, NE). Antibodies to AOX
(Elthon et al., 1989), TIM17 (Murcha et al., 2005), and UCP (Considine
et al., 2001) have been described previously. For the immunodetection
experiments, mitochondrial proteins were isolated from the same water
culture-grown seedling tissue that was used to prepare mitochondria for
the in vitro import experiments, except for metaxin plants, for which tissue
was always obtained from aerial rosettes.
IgG was purified for pull-down and inhibition studies using the Pierce
Melon IgG purification kit according to the manufacturer’s instructions
(Pierce). For the pull-down assays, TOM20, mtOM64, and METAXIN were
expressed in the wheat germ RTS (Roche), and on completion of the 24-h
synthesis reaction, 1 mL of freshly synthesized radiolabeled precursor
was incubated with 15 mL of freshly synthesized TOM20, mtOM64, or
METAXIN and incubated at 248C for 1 h. RTS lysate programmed to
synthesize GUS was used as a control, antibody obtained from Sigma-
Aldrich. At the end of the incubation period, the volume was adjusted to
300 mL in PBS with 1% (w/v) BSA, and 50 mL of Protein A Sepharose 4B
conjugate (Sigma-Aldrich) was added to preclear the lysate for any
nonspecific binding to the Protein A Sepharose (Sambrook et al., 1989).
After incubation with gentle mixing for 1 h, the Protein A Sepharose was
removed by centrifugation. Five microliters of the appropriate purified
antibody was added and incubated for 1 h, followed by addition of 50 mL
of Protein A Sepharose and incubation for a further 1 h. The beads were
pelleted by centrifugation at 5000 rpm, washed in 200 mL of PBS with 1%
(w/v) BSA, and repelleted. Products were analyzed by SDS-PAGE
followed by exposure to a BAS-TR2040 plate for 48 h and imaged in a
BAS2500 (Fuji). Four different sets of negative controls were performed to
ensure that the antibody was only pulling down the target proteins and
that interactions of the precursor protein with the test proteins was
mediated by the mitochondrial targeting signal. First, AOX and FAd
proteins were engineered that lacked the mitochondrial targeting signal,
called AOXDp and FAdDp, respectively (see below for details), to test if
interaction was with this region of the protein. Second, the pull downs
were performed in the absence of TOM20, METAXIN, mtOM64, or GUS to
ensure that any radiolabeled precursor was not being pulled down
directly with the antibody. Third, the nonmitochondrial protein GUS was
used as a comparison. Finally, the specificity of the pull down was tested
by protein gel blotting.
Yeast Two-Hybrid Interaction Assay
The Clontech Matchmaker two-hybrid system was used to determine
interactions between the precursor proteins AOX, AOXDp, FAd, FAdDp,
PiC, GR, and TOM40 with TOM20-2, TOM20-3, TOM20-4, mtOM64,
METAXIN, and At TOC64-III. The latter were cloned into the bait vector
pGADT7-Rec[2] (Leu selection) and the precursor proteins cloned into the
prey vector pGBKT7 (Trp selection) by recombination cloning according
Mitochondrial Preprotein Import 3755
to the manufacturer’s instructions. Positive interactions were screened
via two rounds of selection, first by growth on -His (with -Leu and -Trp)
media and secondly by growth on -Ade media (with -Leu, -Trp, and -His);
this higher stringency protocol reduces the rate of false positives (James
et al., 1996). In the case of the AOX and FAd precursor proteins, the first 42
and 31 amino acids were removed as these regions had been previously
shown to contain the mitochondrial targeting activity (Dessi et al., 2003;
Lee and Whelan, 2004).
BN-PAGE
Mitochondrial membrane complexes were solubilized in 5% (v/v) digito-
nin and separated by first dimension BN-PAGE as described previously
(Eubel et al., 2005). Proteins from the first dimension gel were transferred
onto nitrocellulose membrane and immunodetection performed as de-
scribed above.
Clones/Constructs
The cDNAs encoding the following proteins have been described previ-
ously: AOX (GenBank accession number X68702) (Whelan et al., 1995),
FAd (GenBank accession number X79057) (Dessi et al., 1996), GR
(At3g54660) (Chew et al., 2003a), PiC (GenBank accession number
ABO16064) (Bathgate et al., 1989; Murcha et al., 2004), and RPS10
(At3g22300) (Adams et al., 2002). METAXIN (At2g19080), mtOM64
(At5g09420), At TOC64-III (At3g17970), TOM40 (At3g20000), and VDAC
(At3g01280) were amplified from Arabidopsis cDNA as described previ-
ously (Murcha et al., 2003).
GFP Subcellular Localization
GFP subcellular localization was performed by cloning GFP-5 in frame
with the N or C terminus of the cDNA clone and subsequent transformation
of Arabidopsis suspension cells by biolistic transformation (Thirkettle-
Watts et al., 2003; Lee and Whelan, 2004). RFP was fused to the targeting
signal of soybean (Glycine max) alternative oxidase (AOX-RFP) as a
mitochondrial control (Murcha et al., 2007). Fluorescence patterns were
visualized after 48 h under an Olympus BX61 fluorescence microscope
and imaged using CellR imaging software.
In Vitro Mitochondrial Protein Import
[35S]-Met–labeled precursor proteins were synthesized using rabbit re-
ticulocyte TNT in vitro transcription/translation lysate (Promega) as de-
scribed previously (Whelan et al., 1995). The use of equivalent quantities
of mitochondria from different genotypes in import reactions was ensured
by triplicate measurement of protein concentration with the Coomassie
protein assay reagent (Pierce). Time-course analysis of precursor protein
import into intact mitochondria isolated from wild-type or mutant plants
was performed as described previously (Whelan et al., 1995), but with the
addition of 1 mM GTP and 1 mM NADH to the import master mix. Briefly,
250 mg of mitochondria were added to 450 mL of ice-cold import master
mix (0.3 M sucrose, 50 mM KCl, 10 mM MOPS, 5 mM KH2PO4, 0.1% [w/v]
BSA, 1 mM MgCl2, 1 mM Met, 0.2 mM ADP, 0.75 mM ATP, 5 mM suc-
cinate, 5 mM DTT, 1 mM GTP, and 1 mM NADH, pH 7.5) and incubated on
ice for 3 min. Twenty-five microliters of radiolabeled precursor protein
was added and the import reaction initiated by incubation at 268C with
gentle rocking. One hundred microliters containing 50 mg mitochondrial
protein was removed at 2, 5, 10, and 20 min. Upon removal, each aliquot
was mixed with 3.2 mg PK and incubated on ice for 30 min. Proteolysis
was inhibited by the addition of 1 mL of 100 mM PMSF. Mitochondria were
reisolated by centrifugation at 20,000g for 3 min at 48C, the mitochondrial
pellet resuspended in sample buffer, and the protein sample separated by
SDS-PAGE gel and imported radiolabeled proteins detected as outlined
previously (Murcha et al., 1999). PK protected mature radiolabeled pro-
tein was quantitated at each time point and normalized to the highest time
point measurement for replicate experiments (n ¼ 3 6 SE).
For import competition assays, competitor proteins were synthesized
using the RTS wheat germ lysate in vitro transcription/translation system
(Roche), according to the manufacturer’s instructions. Competitor pro-
tein was used in the wheat germ lysate mix (consisting of wheat germ
lysate resuspended in wheat germ reconstitution buffer) in which it was
synthesized, without further purification from the wheat germ lysate mix.
Five microliters of radiolabeled precursor protein was mixed with 15 mL
of competitor protein (2 mg) in wheat germ lysate mix and preincubated
at room temperature for 15 min. A control competition reaction was
performed by preincubation of precursor protein with 15 mL (2 mg) of GUS
in wheat germ lysate mix. Therefore, the same quantity of wheat germ
lysate mix was added to both competitor and control competition import
reactions. The expressed competitor protein was quantitated by sepa-
ration of the proteins by SDS-PAGE and protein gel blotting with Anti-6-
His antibodies, as all proteins expressed in this system contained a 6-His
tag. The radiolabeled precursor/competitor solution was added to an
import master mix containing mitochondria and incubated at 268C for 10
min, after which the import reaction was stopped by incubation on ice and
PK treatment. PK-protected mature radiolabeled protein was quantitated
at each time point and normalized to the amount of protein imported after
preincubation with GUS. To test for the effect of the wheat germ lysate
mix upon mitochondrial protein import, AOX and FAd precursor proteins
were preincubated for 15 min at room temperature with either 15 mL of
wheat germ lysate mix or 15 mL of wheat germ reconstitution buffer alone,
after which protein import was performed as described above. Mature
imported radiolabeled protein was quantitated after import and normal-
ized to the amount of protein imported in a control import reaction in
which precursors had not been preincubated in wheat germ lysate mix or
wheat germ reconstitution buffer.
PK Digestion of Mitochondrial Outer Membrane Proteins
Mitochondria were isolated from liquid grown plants as described above
and 500 mg of mitochondria incubated with 0 to 32 mg PK in a final volume
of 300 mL of 13 wash buffer (Day et al., 1985) on ice for 15 min. Proteolysis
was inhibited by the addition of 2 mL of 100 mM PMSF. Mitochondria were
reisolated by centrifugation at 20,000g for 5 min and resuspended in
SDS-PAGE sample buffer. Fifty micrograms was separated in each lane
by SDS-PAGE and immunodetection with specific antibodies performed.
Two-Dimensional IEF/SDS-PAGE
Mitochondria for two-dimensional IEF/SDS PAGE separation were further
purified by centrifugation on a 45% Percoll gradient in 13 wash buffer
minus BSA (Day et al., 1985). The mitochondrial fraction was washed
twice in 13 wash buffer minus BSA, then pelleted by centrifugation at
31,000g for 15 min. Mitochondrial proteins were separated by two-
dimensional IEF/SDS-PAGE as described previously (Ito et al., 2006).
Proteomic Analysis of Mitochondrial Proteins by
Mass Spectrometry
Trypsin digestion and tandem mass spectrometry identification of pro-
teins from two-dimensional PAGE gels was performed as described
previously (Chew et al., 2003a).
Transcript Analysis
Quantitative RT-PCR was performed as per the manufacturer’s instructions
using the Roche LC480 and LightCycler480 SYBR Green I Master (Roche)
3756 The Plant Cell
in a total volume of 10 mL. Gene-specific primers were used with cDNA
pools synthesized from wild-type and metaxin rosette leaf total RNA as
described previously (Murcha et al., 2003). Primers for measurement of
transcript abundance are listed in Supplemental Table 1 online. Transcript
abundance for each amplicon was normalized to the wild-type sample.
Phylogenetic Analysis
The phylogenetic relationship was inferred using the neighbor-joining
method. Sequence alignment was performed using ClustalW (see Sup-
plemental Figure 5 online). The bootstrap consensus tree inferred from
5000 replicates was taken to represent the relatedness of the sequences
analyzed. Branches corresponding to partitions reproduced in <50%
bootstrap replicates are collapsed. The percentage of replicate trees in
which the associated sequences clustered together in the bootstrap test
(5000 replicates) are shown next to the branches. The tree is drawn to
scale, with branch lengths in the same units as those of the evolutionary
distances used to infer the phylogenetic tree. All positions containing
gaps and missing data were eliminated from the data set (complete
deletion option). There were a total of 263 positions in the final data set.
Phylogenetic analyses were conducted in MEGA4.
Accession Numbers
Sequence data from this article can be found in the National Center for
Biotechnology Information and Arabidopsis Genome Initiative databases
under the following accession numbers: TOM20-1, NP_189343 and
At3g27070; TOM20-2, NP_174059 and At1g27390; TOM20-3,
NP_189344 and At3g27080; TOM20-4, NP_198909 and At5g40930;
MEXAXIN, NP_565446 and At2g19080; mtOM64, NP_196504 and
At5g09420; Pic, X57566; AOX, X68702; FAd, X79057; GR, X27456;
TOM40, NP_188634 and At3g2000; VDAC, Q9SMX3 and At5g15090;
ACT2, NM_112764 and At3g18780; UBC, ABF59034 and At5g25760.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Analysis of the Expression of TOM20-1.
Supplemental Figure 2. Null Mutations in Any of the TOM20 Genes
Do Not Lead to Deficiencies in Mitochondrial Protein Import.
Supplemental Figure 3. Effect upon Protein Import by Components
of the Wheat Germ Lysate Expression System.
Supplemental Figure 4. metaxin Mutants Are Depleted in METAXIN
Transcript.
Supplemental Figure 5. ClustalW Protein Sequence Alignment of Plant
METAXIN, Animal METAXIN1, and Fungal SAM37 Protein Sequences.
Supplemental Figure 6. The Mitochondrial Proteome Is Not Signif-
icantly Altered by Lesions in METAXIN, mtOM64, or Any TOM20
Isoform.
Supplemental Figure 7. Negative and Positive Controls for Yeast
Two-Hybrid Assay.
ACKNOWLEDGMENTS
This work was supported by the Australian Research Council Centre of
Excellence Program CEO561495. We thank Harvey Millar and Ian Small
for useful suggestions.
Received January 17, 2007; revised October 8, 2007; accepted October
16, 2007; published November 2, 2007.
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Chapter 6 Arabidopsis Mia40
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Chapter 6
Arabidopsis Mia40
Chapter 6 Arabidopsis Mia40
87
Foreword to Study V Whilst searching for proteins that were potentially dual targeted to mitochondria
and peroxisomes, it was uncovered that the Arabidopsis homolog of the yeast Mia40
contains a PTS1 signal, indicating a peroxisomal location for this protein in plants.
Mia40 is part of an import pathway in the intermembrane space of yeast and humans
(Chacinska et al., 2004). This pathway, termed the mitochondrial import and assembly
pathway (MIA), contains two essential proteins in yeast: Erv1 and Mia40 (Chacinska et
al., 2004; Rissler et al., 2005). The MIA pathway is a disulfide relay system, involved
in forming disulfide bonds into newly imported intermembrane space proteins such as
the small Tims (Sideris and Tokatlidis, 2010). As there was no functional data on the
Arabidopsis Mia40, a study was undertaken to confirm its subcellular location and
function within plant cells.
In contrast to findings in yeast, this study showed that Arabidopsis Mia40 is a
dual targeted protein in plants, targeted to both peroxisomes and the mitochondrial
intermembrane space. The Arabidopsis Mia40 was also found to be non-essential, with
knockout plants displaying no abnormal phenotype. However, at a molecular level, the
absence of Mia40 had a number of interesting phenotypes. The absence of Mia40 in
Arabidopsis led to a decrease in protein levels in both peroxisomes and mitochondria of
the copper/zinc chaperone for superoxide dismutase 1 (Ccs1) and the mitochondrial and
peroxisomal copper/zinc superoxide dismutases (CSD1 and CSD3 respectively). The
loss of Mia40 also led to a decrease in the capacity of complex I of the respiratory chain
and ΔMia40 plants also demonstrated compromised assembly of complex I. While
Mia40 in yeast has been implicated in the import of small Tim proteins (Chacinska et
al., 2004), the absence of Arabidopsis Mia40 led to no change in the import of small
Tim proteins in plants. Thus Arabidopsis Mia40 has taken on new roles in peroxisomes
and mitochondria in plants and the disulfide relay system operates in a mechanistically
different manner in plant mitochondria compared to yeast.
Conserved and Novel Functions for Arabidopsis thalianaMIA40 in Assembly of Proteins in Mitochondria andPeroxisomes□S
Received for publication, March 7, 2010, and in revised form, September 4, 2010 Published, JBC Papers in Press, September 9, 2010, DOI 10.1074/jbc.M110.121202
Chris Carrie‡, Estelle Giraud‡, Owen Duncan‡, Lin Xu‡, Yan Wang‡, Shaobai Huang‡, Rachel Clifton‡,Monika Murcha‡, Aleksandra Filipovska§, Oliver Rackham§, Alice Vrielink¶, and James Whelan‡1
From the ‡Australian Research Council Centre of Excellence in Plant Energy Biology, the §Western Australian Institute for MedicalResearch, Centre for Medical Research, and the ¶School of Biomedical, Biomolecular, and Chemical Sciences, University of WesternAustralia, Crawley, Western Australia 6009, Australia
The disulfide relay system of the mitochondrial intermem-brane space has been extensively characterized in Saccharomy-ces cerevisiae. It contains two essential components, Mia40 andErv1. The genome of Arabidopsis thaliana contains a singlegene for each of these components. Although insertional inacti-vation of Erv1 leads to a lethal phenotype, inactivation ofMia40results in no detectable deleterious phenotype. A. thalianaMia40 is targeted to and accumulates in mitochondria and per-oxisomes. Inactivation of Mia40 results in an alteration of sev-eral proteins in mitochondria, an absence of copper/zinc su-peroxide dismutase (CSD1), the chaperone for superoxidedismutase (Ccs1) that inserts copper into CSD1, and a decreasein capacity and amount of complex I. In peroxisomes theabsence of Mia40 leads to an absence of CSD3 and a decrease inabnormal inflorescencemeristem 1 (Aim1), a�-oxidation path-way enzyme. Inactivation of Mia40 leads to an alteration of thetranscriptome of A. thaliana, with genes encoding peroxisomalproteins, redox functions, and biotic stress significantly chang-ing in abundance. Thus, the mechanistic operation of the mito-chondrial disulfide relay system is different inA. thaliana com-pared with other systems, and Mia40 has taken on new roles inperoxisomes and mitochondria.
Characterization of the proteomes of mitochondria fromSaccharomyces cerevisiae (yeast), mammals, and plants indi-cates that they contain from 1000 proteins in yeast to �2000 inhigher organisms (1). As the coding capacity of mitochondria islimited to between 8 and approximately 50 proteins in yeast andplants, respectively (2), the majority of mitochondrial proteinsare encoded by nuclear located genes, translated in the cytosol,and imported into mitochondria. The import of hundreds ofdifferent proteins is achieved by the combined action of a num-ber of multisubunit, integral membrane protein complexes,known as translocases. These translocases work in conjunctionwith a variety of soluble components located in the cytosol,intermembrane space, andmitochondrialmatrix, such as chap-erones, peptidases, and assembly factors (3, 4).
The outer mitochondrial membrane contains the TOMcomplex (translocase of the outer membrane) and the sortingand assembly machinery; the latter is also known as the TOBcomplex (topogenesis of�-barrel proteins) (4). Almost allmito-chondrial proteins are initially recognized by the outer mem-brane complex and are passed to other components dependingon their final location.�-Barrel proteins of the outermembranesuch as Tom40 and voltage-dependent anion channel protein(VDAC) are passed to the sorting and assembly machinerycomplex (5). Proteins imported via the general and carrierimport pathways are passed to the translocases of the innermembrane 23 (TIM23) and 22 (TIM22), respectively (3, 4). Pro-teins are passed to the TIM23 complex directly from the outermembrane complex with the aid of Tim50 (6, 7) and also pos-sibly Tim23, where the N-terminal region has been shown totransiently associate with the outer membrane (8). Proteins aretransferred to the TIM22 complex via the aid of the small inter-membrane space proteins Tim9 and -10 (3, 4). A number of vari-ations of thesemain pathways can occur. These include the cross-ing over between pathways (9) and the utilization of differentsorting processes. Proteins can be either stop-transfer-sorted orconservative-sorted, with the latter pathway using the Oxa1ptranslocase located on the innermitochondrialmembrane (10). Avariety of other protein import pathways also exist, such as thoseutilized for the import of cytochrome c (11) and the small Timproteins of the intermembrane space (12, 13).The import pathway of the small intermembrane space pro-
teins, such as Tim8, -9, -10, and -13, is the most recentlydescribed protein import pathway. Their import is achieved viathe mitochondrial intermembrane space assembly machinery(MIA)2 that consists of Mia40 and Erv1 (3, 12, 14), both ofwhich are essential proteins in yeast (15, 16). Characterizationof the import pathway of Tim9 and -10 in yeast revealed that aslittle as nine amino acids are required to achieve transfer fromthe outer membrane complex on the outer membrane toMia40, which acts as a receptor in the intermembrane space(17, 18). These proteins then undergo oxidative folding in theintermembrane space. Conserved cysteine residues in these
□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Tables 1 and 2 and Figs. 1– 4.
1 To whom correspondence should be addressed: ARC COE PEB, University ofWestern Australia, 35 Stirling Highway, Crawley WA 6009, Australia. Tel.:61-8-64881749; Fax: 61-8-64884401; E-mail: [email protected].
2 The abbreviations used are: MIA, mitochondrial intermembrane assembly;CA2, carbonic anhydrase 2; Kat2, 3-ketoacyl-CoA thiolase; Aim1, alteredinflorescence meristem 1; Sod1, superoxide dismutase 1; Ccs1, copperchaperone for CSD1; RFP, red fluorescent protein; BN, blue native; TES,2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 46, pp. 36138 –36148, November 12, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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proteins are oxidized byMia40, which is subsequently oxidizedby Erv1. Erv1 is oxidized by cytochrome c, which is in turnoxidized by cytochrome c oxidase, with molecular oxygen act-ing as the final electron acceptor (19).As mitochondrial endosymbiosis occurred only once in evo-
lutionary history (2), many features of mitochondrial biologyare conserved across wide phylogenetic gaps. With respect toprotein import into mitochondria, it is observed that whereaspore or channel-forming subunits of the membrane-boundtranslocases are well conserved, variability is often seen withother components of the import apparatus (20, 21). Thus, com-parison of the plant import apparatus to that of yeast (andmammalian) systems reveals that although Tom40, Sam50,Tim17 and -23, Tim22, and Oxa1p are well conserved, othercomponents differ to varying extents. Thus, none of the threefunctionally characterized protein import receptors of plantmitochondria is orthologous to yeast or mammalian proteinimport receptors (22). Furthermore, the mitochondrial pro-cessing peptidase is membrane-bound in plants compared withmatrix located in yeast (23). In contrast, the presequencedegrading peptidase is located in the matrix in plants, whereasits orthologous counterpart in yeast is located in the intermem-brane space (24, 25).To date no studies have been carried out on components of
the MIA import pathway in plants. As an essential pathwayin yeast, it might be expected to function in a similar manner inplants. We have analyzed the function of Mia40 and Erv1 inArabidopsis thaliana with the aim of defining the essentialcomponents of theMIA pathway, their location, and the effectsof inactivation.
EXPERIMENTAL PROCEDURES
cDNA Clones and Constructs—The chromosomal loci forAtMia40 (At5g23395) and AtErv1 (At1g49880) have been pre-viously identified (26). TheOryza sativa (rice)Mia40was iden-tified as Os04g44550 by using AtMia40 to search the ricegenome (27). cDNAs of AtMia40, AtErv1, and OsMia40 werecloned using gateway cloning (Invitrogen) into vectors express-ing GFP as either N- or C-terminal fusions under the controlof a constitutive promoter (28). The human Mia40 cDNA(AAH33775) was cloned as both N- and C-terminal fusionswithGFP into pcDNA3 (Invitrogen) under the control of a con-stitutive promoter using standard cloning techniques (29, 30).The full cDNA of A. thaliana Tim9 (At3g46560), copper chap-erone for CSD1 (Ccs1, At1g12520), copper/zinc superoxidedismutase 1 (CSD1, At1g08830), copper/zinc superoxide dis-mutase 3 (CSD3, At5g18100), and carbonic anhydrase 2 (CA2,At1g47260) were cloned into pDest14 (Invitrogen) using gate-way-cloning techniques (Invitrogen) for in vitro transcriptionand translation. The cDNA clones encoding the following pro-teins have been described previously: AOX (X68702) (31), Pic(ABO16064) (32, 33), and NDC1 (At5g08740) (34).GFP Subcellular Localizations—To determine the subcellu-
lar localization of AtMia40 and OsMia40 A. thaliana cell cul-ture was transformed by biolistic transformation as previouslydescribed (28). GFP and RFP expression and targeting werevisualized using a BX61 Olympus microscope (Olympus) usingexcitation wavelengths of 460/480 nm (GFP) and 535/555 nm
(RFP) and emission wavelengths of 495–540 nm (GFP) and570–625 nm (RFP). Subsequent images were captured usingCell imaging software as previously described (28). For theHsMia40, 143B osteosarcoma cells were plated onto 13-mmdiameter glass coverslips and allowed to attach overnight.Cells were transfected using FuGENEHDwith both GFP andRFP plasmids for 48 h and washed with Tris-buffered saline(5 mM Tris/HCl (pH 7.4), 20 mM NaCl). Cells were mountedin DABCO (1,4-diazabicyclo(2.2.2)octane)/polyvinyl alco-hol medium. Images were acquired using an Olympus DP70fluorescent inverted microscope as previously described (29).T-DNA Insertion Lines—The following T-DNA insertion
lines were obtained from the SALK collection (35) and geno-typed by PCR to confirm homozygosity for the T-DNA in-sert: AtMia40 (At5g23395): SALK_044358 and AtErv1(At1g498890): SALK_110883, SALK_131166, SALK_001649.Organelle Purification—Mitochondria for in vitro import
experiments were harvested from 20 g (fresh weight) of 14-day-old A. thaliana seedlings grown in liquid culture as previouslydescribed (22) with the BSA omitted from the last two washsteps. Typically 2–3mg ofmitochondrial protein was obtained.For immunodetection assays, highly purifiedmitochondria andperoxisomes were purified from 7-day-old A. thaliana cell sus-pension using free flow electrophoresis as described by (36).In Vitro Import Studies—[35S]Met-labeled precursor pro-
teins were synthesized using rabbit reticulocyte TNT in vitrotranscription/translation lysate (Promega, Melbourne, Austra-lia) as described previously (37). The use of equivalent quanti-ties of mitochondria from different genotypes in import reac-tions was ensured by triplicate measurement of proteinconcentration with the Coomassie protein assay reagent(Thermo Scientific, Rockford, IL). Time course analysis of pre-cursor protein import into intact mitochondria isolated fromwild type (Col-0) or mutant plants was performed as describedpreviously (22, 37). Proteinase K protected, mature, radiola-beled protein was quantified at each time point and normalizedto the highest time point measurement for replicate experi-ments (22).BN-PAGE Imports—For in vitro imports analyzed on BN-
PAGE gels, import assays were performed as for SDS-PAGEexcept 250 �g of mitochondria were used per time point. Afterthe import was carried out for the required time, mitochondriawere pelleted at 20,000 � g for 5 min and then subjected toBN-PAGE according to the method described by Jansch et al.(38). Mitochondrial proteins (250 �g) were solubilized with 5%(w/v) digitonin in a buffer containing 30 mM HEPES, 150 mM
potassium acetate, and 10% (v/v) glycerol (pH 7.4) and incu-bated on ice for 15min. Samples were centrifuged for 20min at15,000 � g, and Serva Blue G (0.2% (v/v) final) was added to thesupernatant. Samples were loaded onto a 4.5–16% (v/v) gradi-ent gel. After migration, gels were fixed in 40% (v/v) methanol,10% (v/v) acetic acid, dried, and exposed as per SDS-PAGE gels.Immunodetection of Proteins—Mitochondria and peroxi-
somes (25 �g) were resolved by SDS-PAGE, transferred toHybond-C extra nitrocellulose membrane, and immunode-tected as previously outlined (39). To generate antibodiesto AtMia40, AtErv1, AtTim23-2 (At1g72750), AtSam50(At3g11070), and the Rieske iron sulfur protein (RISP,
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At5g13430), recombinant proteins containing the full-lengthAtMia40 and AtErv1, amino acids 31–144 for AtTim23-2, thefirst 200 amino acids of AtSam50, and amino acids 21–151 ofRieske iron sulfur protein fused to an N-terminal His6 affinitypurification tag were expressed in Escherichia coli strain BL21(DE3)pLys (Stratagene, La Jolla, CA). The recombinant proteinwas purified by denaturing immobilizedmetal affinity chroma-tography (IMAC) using the Bio-Rad Profinia protein purifica-tion system. The resultant eluate was separated by SDS-PAGE,and the recombinant protein was extracted using a Bio-RadModel 422 Electro-Eluter. Buffer exchange was performedusing an Amicon Ultracel 5K centrifugal filter device (Milli-pore, Sydney, Australia) such that the antigen was re-sus-pended in PBS solution, recovering a total of 1 mg (AtErv1), 2mg (AtMia40), 2mg (AtTim23-2), 3mg (AtSam50), and 3mgofRieske iron sulfur protein for inoculation. Four separate doseswere administered to a rabbit at regular intervals over a3-month period using standard protocols and Freund’s com-plete adjuvant solution (40). Other antibodies used in this studyhave been described previously: 3-ketoacyl-CoA thiolase (Kat2)(41), Tim17-2 (42), Tom20-2, andTom20-4 (22), Tom20-3, andTom40 (28), coxII, ATP synthase, Ccs1, and CSD1 wereobtained from Agrisera (Vannas), voltage-dependent anionchannel protein (VDAC; PM035) andE1� of pyruvate dehydro-genase (PM030) were obtained from Dr. Tom Elthon (Univer-sity of Nebraska, Lincoln, NE), alternative oxidase (43), andNAD9 (44). The altered inflorescence meristem 1 (Aim1) anti-body was obtained from Dr. Douglas Muench (University ofCalgary).Complex I Activity Assays—Complex I activity wasmeasured
on 140 �g of mitochondria in 1 ml of respiration buffer (0.3 M
sucrose, 5mMKH2PO4, 10mMTES, 10mMNaCl, 2mMMgSO4,and 0.1% (w/v) BSA (pH 6.8)) obtained after freeze-thawing ofthe mitochondria using a Clark-type oxygen electrode (Han-satch Instrument). 1 mM deamino-NADH was added to thechamber, oxygen consumptionwasmeasured, and 2mMCCCPwas then added to ensure mitochondria were uncoupled. Rote-none (5 mM) was then added to inhibit complex I and to mea-sure the rate of oxygen consumption of the alternative NADHdehydrogenases. Finally 0.1 mM of KCN was used to terminatethe reaction. The activity of complex I was defined as therotenone-sensitive rate of oxygen consumption, determinedby subtracting the rate of oxygen consumption of the alter-native NADH dehydrogenases from the total oxygen rateconsumption.Global Transcript Analysis—Analysis of the changes in tran-
script abundance between Col-0 and �mia40 plants in 18-day-old A. thaliana seedlings was performed using AffymetrixGeneChipTM A. thaliana ATH1 Genome Arrays (Affymetrix,Santa Clara, CA). Green tissue from three seedlings was pooledfor each biological replicate; Col-0 and �mia40 tissue sampleswere collected in biological triplicate. For each replicate, totalRNA was isolated from the leaves using the RNeasy Plant MiniProtocol (Qiagen, Clifton Hill, Australia) and quality-verifiedusing a Bioanalyzer (Agilent Technologies, Palo Alto, CA), andspectrophotometric analysis was carried out to determine theA260:A280 and A260:A230 ratios. Preparation of labeled aRNAfrom 500 ng of total RNA (3� IVT Express kit, Affymetrix) and
target hybridization as well as washing, staining, and scanningof the arrays was carried out exactly as described in theAffymetrix GeneChipTM Expression Analysis Technical Man-ual using an Affymetrix GeneChip Hybridization Oven 640, anAffymetrix Fluidics Station 450, and an GeneChip Scanner3000 7G at the appropriate steps.Statistical Analysis—Data quality was assessed using GCOS
1.4 before CEL files were exported into AVADIS Prophetic(Version 4.3, Strand Genomics, San Francisco, CA) and PartekGenomics Suite software, Version 6.3 (Partek, St. Louis, MO)for further analysis. MAS5 normalization algorithms were car-ried out to generate present/absent calls across the arrays. Onlythose probe sets that were called present in at least two of threereplicates in at least one genotype were included for furtheranalysis. Ambiguous probe sets and bacterial controls wereremoved, resulting in a final data set of 12,551 probe identifiers.CEL files were also subjected to guanosine cytosine robustmulti-array average normalization. Correlation plots wereexamined between all arrays using the scatter plot function; inall cases r � 0.97 (data not shown). Guanosine cytosine robustmulti-array average-normalized gene expression values wereanalyzed to identify differentially expressed genes by a regular-ized t test based on a Bayesian statistical framework using thesoftware program Cyber-T (45). Cyber-T employs a mixturemodel-based methods described by Allison et al. (46) for thecomputation of the global false positive and false negative levelsinherent in a DNA microarray experiment. The rates of falsepositives and false negatives as well as true positives and truenegatives at any given p value threshold are estimated, i.e. aposterior probability of differential expression (PPDE) (p) valuefor each genemeasurement and a PPDE (�p) value at any givenp value threshold based on the experiment-wide global falsepositive level and the p value exhibited by that gene. There were322 unique transcripts that were identified as significantly dif-ferentially expressed in the �mia40 plants compared withCol-0 after false discovery rate correction at PPDE (�p) �0.95(95% confidence interval) (supplemental Table 2). Functionalcategorization using GO-biological process annotations wasperformed on the total present set (12,551 transcripts) alongwith the 322 transcripts defined as differentially expressed(either positively or negatively). GO annotations (biologicalprocess) were obtained from the TAIR webpage. To determineany changes in distributions of different cellular locations, tran-scripts in the total present set and differentially expressed setwere annotated based on their subcellular location: plastid,mitochondrial, peroxisomal, or other. Lists of genes encodingplastid and peroxisomal proteins were generated using theSUBA data base and included genes based on experimentaldetermination (found by mass spectrometry or GFP profiling).The list ofmitochondrial proteins used here has been describedpreviously (47). The percentage distribution of each categorywas compared with that of the total present set using a �2 test,and percentile distributions were considered to be significantlydifferent at a 98% confidence interval. To gain a qualitativeoverview of changes in transcript abundance for�mia40 plantscompared with Col-0, the MapMan software was used (48).Only transcripts with significant changes after false discoveryrate were displayed.
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RESULTS
Erv1 Is an Essential Protein in A. thaliana, but Mia40 IsDispensable—To determine the role of Mia40 and Erv1 inA. thaliana, T-DNA insertion lines that disrupt the genes and,thus, do not produce functional proteins were screened. In thecase of Mia40, one line with a T-DNA insert that disrupted thegene was obtained from screening all lines annotated as havinginsertions in Mia40 (�mia40) (Fig. 1A). The absence of theproteinwas confirmedbyWestern blot analysis against purifiedmitochondria from Col-0 and �mia40 plants (see Fig. 3A). Thelack of Mia40 had no discernable effect on growth (Fig. 1A),with normal seed set production observed compared withCol-0 growth under identical conditions. Even though a singleinsertional mutant could only be obtained for Mia40, theabsence of the protein was confirmed byWestern blotting, andit is the lack of expected phenotype that is reported. Further-
more, backcrossing and Southern blot analysis indicate a singleinsertion in the line characterized.In contrast, despite screening three T-DNA lines for Erv1, no
homozygous T-DNA-inactivated lines for Erv1 could beobtained. In addition to back-crossing the T-DNA lines toremove any other T-DNA inserts or mutations that may bepresent, analysis of the seed from self- fertilized heterozygousplants (Erv1/erv1-T-DNA) consistently resulted in 25% of theseed being aborted for all lines analyzed (Fig. 1B). This is con-sistent with a lethal phenotype due to the absence of a func-tional gene encoding this protein.The lethality of �erv1 was not surprising as it is an essential
protein in yeast. In yeast it is required for the import and assem-bly of small intermembrane space proteins (16, 49), specificallythe small Tims that in turn are required for the import of carrierproteins to the inner membrane. They also play a role in �-bar-rel protein assembly in the outer membrane (3, 50). Therefore,a similar phenotype would be predicted for �mia40 plantsbased on extensive studies in yeast andmammalian systems (12,15, 51, 52). Examination of the sequences for both predictedproteins in A. thaliana revealed that they differed comparedwith their yeast orthologues. In the case ofA. thalianaMia40 itwas a much smaller protein of 162 amino acids (supplementalFig. 1A). ThemammalianMia40 protein is also shorter than theyeast orthologue, and the smaller “core” conserved region ofMia40 has been shown to be functional in the disulfide relaysystem (53). The only noticeable “unique”feature of theA. thaliana Mia40 was the presence of a putative peroxisomalPTS1 targeting signal, SKL, at theC terminus (54). Examinationof the A. thaliana Erv1 protein sequence revealed that it dif-fered in its arrangement of cysteine pairs to that of yeast andhumans (supplemental Fig. 1, B and C). In all Erv1 sequencesanalyzed to date two pairs of cysteinemotifs are conserved in allorganisms, the CXXC and CX16C motifs. In regard to the thirdcysteine pair, yeast and human Erv1 proteins have an N-termi-nal CXXCmotif, whereas A. thaliana has a C-terminal-locatedCXXXXC motif (Fig. 1C) (55). This cysteine motif shows dis-tinct similarities to the yeast endoplasmic reticulum Erv2p (56)and the trypansome Erv1, both of which also have their thirdcysteine pair at theC terminus (Fig. 1C) (57). Phylogenetic anal-ysis of all Erv1 and Mia40 protein sequences revealed that theplant proteins formed distinct groups (supplemental Fig. 2),noticeably the trypanosome Erv1 branches closest to the plantgroup, this group being the only group containing the thirdcysteine pair at the C-terminal end of the protein (Fig. 1C, sup-plemental Fig. 2).Mia40 Is Targeted to Mitochondria and Peroxisomes in
Plants—We fused A. thaliana Mia40 to GFP at its N and Ctermini to determine whether the predicted peroxisomal PTS1targeting signal was functional. When GFP was fused to the Nterminus, a pattern identical to peroxisomal-targeted RFP wasseen (Fig. 2A,GFP-AtMIA40). AsMia40 has been characterizedas an exclusively mitochondrial protein in yeast and mamma-lian systems (organisms that also contain peroxisomes), it wasinvestigated if the peroxisomal targeting ability of A. thalianaMia40 was a general feature of plant Mia40 proteins by testingthe targeting of the riceMia40. A similar result was observed in
FIGURE 1. T-DNA insertional inactivation of A. thaliana Mia40 and Erv1.A, shown is a picture of 25-day-old Col-0 and �mia40 A. thaliana plants.B, shown is a representative picture of seeds from Col-0 and �erv1 (heterozy-gous) self-fertilized plants. Arrows indicate aborted seed. C, shown is a sche-matic diagram of Erv1 proteins from different organisms. The gray regionrepresents the most conserved region between different Erv1 sequencescontaining the redox center, the FAD binding site, and two conserved cys-teine domains (CXXC and CX16C ). The location of the final cysteine pair differsbetween organisms. Yeast Erv1 and human Erv1 have it at the N terminus, andthose for yeast Erv2, A. thaliana, and trypanosomes are at the C terminus.Yeast, S. cerevisiae; human, Homo sapiens; Arabidopsis, A. thaliana; trypano-somes, Trypanosoma brucei.
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that rice Mia40 was targeted to peroxisomes when GFP wasplaced at its N terminus (Fig. 2A, GFP-OsMIA40).When GFP was fused to the C terminus of both A. thaliana
and rice Mia40, a mitochondrial pattern of florescence wasobserved (Fig. 2A, top panel). Noticeably, the fluorescence pro-duced ring like structures, which we have previously observedfor mitochondrial proteins linked to GFP (22). They may rep-resent GFP that has been targeted to mitochondria but nottranslocated across the outer membrane, possibly due to thelack of any pulling force by an intermembrane space protein
such as Mia40. High backgroundfluorescence in the cell cytosol wasalways evident with both A. thali-ana and rice Mia40 with GFP fusedto the C terminus despite testing inseveral cell types including onion,A. thaliana leaf, and root (data notshown). This high background fluo-rescence is not observed for a vari-ety of other mitochondrial targetingsignals and is unlikely to be a tech-nical limitation of the system (28,39). Additionally, the subcellularlocalization of Mia40 to both mito-chondria and peroxisomes was con-firmed using Western blotting onhighly purified mitochondrial andperoxisomal fractions using anantibody raised against A. thalianaMia40 (Fig. 2B). This antibodydetected a specific protein in bothhighly purified mitochondria andperoxisomes purified by free flowelectrophoresis (36). No Tim17-2protein or E1�-subunit of pyruvatedehydrogenease (E1�-PDH)wasde-tected in peroxisomes, indicatingthe purity of the peroxisomal frac-tions, thus verifying this novel per-oxisomal location for Mia40 (Fig.2B). The absence of significantperoxisome contamination in themitochondrial fraction was con-firmed using two peroxisomalmarkers, Kat2 and Aim1. Bothmarkers show that the mitochon-drial fraction was free of anysignificant peroxisome contami-nation. As A. thaliana Mia40 ap-peared to target GFP poorly tomitochondria, we investigated theimport of A. thaliana Mia40 intoisolated mitochondria (Fig. 2C).A. thaliana Mia40 was importedinto a protease-protected locationin a membrane potential-indepen-dent manner (Fig. 2C, lanes 1–5),consistent with a location in the
intermembrane space. Rupture of the outer membranebefore adding protease resulted in digestion of importedMia40 (Fig. 2C, lanes 6–9). Import of A. thaliana Tim23 as acontrol verified that the membrane potential was collapsedas import of Tim23 was inhibited in the presence of valino-mycin (Fig. 2C, lanes 1–5) and that the inner membrane wasintact, as evidenced by the presence of a characteristic mem-brane-protected fragment of Tim23 upon the addition ofprotease when the outer membrane was ruptured (Fig. 2C,lanes 6–10).
FIGURE 2. Determination of the targeting ability of Mia40 using GFP tagging, Western blots, and in vitroimport assays. A, the localization of A. thaliana, rice, and human Mia40 was determined by fusing them to GFPat their N and C termini. A. thaliana and rice Mia40 GFP plasmids were transformed into A. thaliana suspensioncells, and human Mia40 GFP plasmids were transfected into 143B osteosarcoma cells. For the A. thalianasuspension cell cultures, the RFP was targeted to mitochondria using the mitochondrial alternative oxidasetargeting signal as a mitochondrial marker and RFP targeted to peroxisomes using a C-terminal SRL sequenceas a peroxisomal marker. For the 143B osteosarcoma cells, RFP was targeted to mitochondria using the mito-chondrial targeting signal from yeast cytochrome c oxidase 4 as a mitochondrial marker and RFP targeted toperoxisomes using a C-terminal SRL sequence as peroxisomal marker. B, Western blot analysis is shown ofisolated mitochondria and peroxisomes with antibodies raised against A. thaliana Mia40, two peroxisomalmarkers, Kat2 and AIM1, and two mitochondrial markers, Tim17-2 and E1� of pyruvate dehydrogenase (E1�-PDH). C, in vitro import of radiolabeled Mia40 into isolated mitochondria is shown. Lane 1, precursor proteinalone. Lane 2, precursor protein incubated with mitochondria under the conditions that support import intomitochondria. Lane 3, as lane 2 with proteinase K added after the incubation of precursor with mitochondria.Lanes 4 and 5, as lanes 2 and 3 with valinomycin added to the import assay before the addition of precursorprotein. Lanes 6 –9, as lanes 2–5 except that the mitochondrial outer membrane was ruptured after the incu-bation period with precursor protein but before the addition of proteinase K. Mit, mitochondria; Mit-OM,mitochondria with the outer membrane ruptured; PK, proteinase K; Val, valinomycin; p, precursor protein band;m*, inner membrane protected fragment of Tim23.
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Analysis of humanMia40 showed that it was only targeted tomitochondria when GFP was fused to the C-terminal end ofhuman Mia40 (Fig. 2A, HsMIA40 GFP). Fusing GFP to theN-terminal end of human Mia40 resulted in no targeting ofGFP as evidence by fluorescence throughout the cytoplasm(Fig. 2A,GFPHsMIA40). Notably, analysis of the targeting abil-ity of A. thaliana Erv1 revealed that it is only targeted to mito-chondria (data not shown).
Cellular Affects of Inactivation of�mia40 inA. thaliana—AsA. thali-ana Mia40 is not an essential pro-tein in A. thaliana, we investigatedthe abundance of variousmitochon-drial and peroxisomal proteins togain insight into the function(s) ofMia40 (Fig. 3). Analysis of the abun-dance of a variety of protein importcomponents in �mia40 plants re-vealed that Erv1 and Tim23-2 hadincreased in abundance, but manyother components were unchanged(Fig. 3A). Examination of a variety ofother mitochondrial proteins re-vealed that although many wereessentially unchanged in abun-dance, there were notable excep-tions (Fig. 3B). First, there was a lackof CSD1, the intermembrane spacelocated copper/zinc superoxide dis-mutase (58, 59), and the chaperoneprotein associatedwithCSD1, Ccs1,which plays a role in inserting cop-per into the active site of CSD1 (58,59). The Nad9 subunit of complex Ialso decreased in abundance signif-icantly (40%). The reduced amountof Nad9 on SDS-PAGE gels wasconfirmed by BN-PAGE (Fig. 3C),which indicated that the reductionof the amount of Nad9was linked toa reduction in the amount of com-plex I in�mia40mitochondria. Thecapacity of complex I was measuredusing deamino NADH with mito-chondria that had been subjected tofreezing and thawing to allowdeamino-NADH access to the ma-trix side of the inner membrane inthe presence of CCCP to ensureunrestricted flow of electrons. Therotenone-sensitive deamino NADH-dependent oxygen consumption was13.9nmol ofO2/min/mgof protein inmitochondria from wild type plantscompared with 6.1 nmol of O2/min/mg of protein in mitochondriafrom �mia40 plants. This indicatedthat complex 1 capacity was reduced
by 44% in mitochondria from �mia40 plants, consistent with thepercentage reduction of the Nad9 protein from complex I.The availability of antibodies to peroxisomal proteins in plants
is limited.NeverthelessWesternblot analysis using three antibod-ies revealed that two proteinswere altered in abundance (Fig. 3D).Although levels of Kat2were unchanged in abundance, CSD3wascompletely absent in peroxisomes from �mia40 plants, whereasAim1 was reduced in abundance by 25%.
FIGURE 3. Western blot analysis to determine the affect of deleting A. thaliana Mia40 on mitochondrialand peroxisomal proteins and blue native gel analysis of �mia40 mitochondria. A, Western blot analysisof mitochondria isolated from Col-0 and �mia40 plants with a variety of antibodies raised against mitochon-drial import components is shown. Numbers on the side represent the relative abundance of the protein in the�mia40 mitochondria expressed relative to Col-0 (1); S.E. is also shown for three biological replicates. B, West-ern blot analysis of mitochondrial proteins is not involved in protein import. VDAC, voltage-dependent anionchannel protein; AOX, alternative oxidase; E1�-PDH, E1�-subunit of pyruvate dehydrogenease; RISP, Rieskeiron sulfur protein. C, blue native gel analysis of Col-0 and �mia40 mitochondria is shown. Mitochondrialmembrane complexes were separated by BN-PAGE and transferred onto a PVDF membrane. Membranes werestained in Coomassie and probed with antibodies against Tom40 and NAD9. D, shown is Western blot analysisof peroxisomes isolated from Col-0 and �mia40 plants with a selection of antibodies raised against peroxi-somal proteins.
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Analysis of Protein Import into Mitochondria of �mia40Plants—To investigate if the changes observed in the amount ofmitochondrial proteins were due to changes in the rate ofimport or stability of imported proteins, in vitro import assayswere carried out with a number of mitochondrial proteins intomitochondria isolated from wild type and �mia40 plants (Fig.4). A variety of precursor proteins were used; the alternativeoxidase, alternative NAD(P)H dehydrogenase (NDC1), a sub-unit of complex 1 that has been labeled as CA2 (60, 61) alongwith CSD1 and Ccs1. Tim9 was used to test the uptake of smallTim proteins into the intermembrane space (17). Finally, weanalyzed the import of the phosphate carrier (Pic), which isimported via the carrier import pathway utilizing the small Timproteins of the intermembrane space (3, 4). Although theuptake of alternative oxidase and NDC1 was reduced to �50%compared with wild type, the import of CSD1 and CA2 wasunaffected, with the import of the former slightly higher in
mitochondria from �mia40 plants.Import of Ccs1 was reduced by 20%in mitochondria from �mia40plants compared with wild type.Thus, although the abundance ofsome proteins was reduced, this didnot result from alternations the rateof import. Note that as Nad9 is amitochondrially encoded subunit inA. thaliana (62), its rate of importcould not be tested.The import of both Tim9 and
Pic were unaffected (Fig. 4, A andB). The latter two proteins wouldbe expected to be affected by�mia40 A. thaliana mitochondriaas Tim9 is a direct substrate ofMia40 in yeast and the import ofcarrier proteins (Pic) depends onthe function of small Tim pro-teins. It has been previously dem-onstrated in plants that smallintermembrane space proteins areinvolved in the import of carrierproteins (22). Thus, the lack of aphenotype for �mia40 plants andthe lack of an effect on proteinimport via the carrier importpathway suggests that Mia40 inA. thaliana does not play an essen-tial role in the disulfide relay systemfor small Tim proteins as observedin yeast.The reduction in the amounts of
the proteins outlined above can beexplained by either a failure to cor-rectly assemble these proteins, al-tered expression of the genes thatencode these proteins, or a combi-nation of both. To investigate bothof these possibilities, the assembly
of proteins into mitochondria after import was investigated(Fig. 4C), and alterations in transcript abundance were ana-lyzed (Fig. 5).To investigate the assembly of protein after import, the
assembly of a complex I protein was analyzed. Assembly ofimported CA2 into complex I was investigated using BN-PAGE to assess the location of newly imported radiolabeledproteins, as previously carried out for a number of studiesinvestigating assembly into multisubunit protein complexesin mitochondria (63, 64). It was evident that incorporation ofCA2 into complex I, especially the supercomplex of complexI and III, was reduced in mitochondria from �mia40 plantscompared with wild type plants (Fig. 4C). In addition toobserving reduced radiolabeling into this higher molecularmass complex, the intensity of radiolabeling at the lowerregions of the gel was also altered (Fig. 4C). Although theintensity of radiolabeling at the lower regions was higher in
FIGURE 4. In vitro uptake assays into mitochondria isolated from Col-0 and �mia40 plants. A, import of thealternative oxidase (mitochondrial (m) and (p) AOX), alternative NAD(P)H dehydrogenase (NDC1), Tim9, phos-phate translocator (Pic), copper/zinc superoxide dismutase 1 (CSD1), Ccs1, and CA2 precursor proteins intoisolated mitochondria is shown. p, precursor protein; m, mature protein, where indicated. B, shown is quanti-fication of the rate of import of the various precursor proteins into mitochondria. The amounts of import intoCol-0 mitochondria after 20 min was set to 1, and all other values are expressed in a relative manner. C, importof the complex I subunit CA2 into Col-0 and �mia40 mitochondria and analyzed by BN-PAGE is shown.
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mitochondria from �mia40 plants compared with wild typeat 10 and 30 min, it was substantially decreased at 120 and180 min. This suggests that it is the assembly of subunits intocomplex I that is affected in mitochondria from �mia40plants compared with wild type plants. Furthermore, theunassembled, imported, radiolabeled CA2 appeared to bedegraded in mitochondria from �mia40 plants as evidencedby the decrease in the intensity of lower molecular massproducts with time (Fig. 4C).
To gain further insight into the effects of deleting Mia40,the transcriptome of �mia40was compared with that of wildtype plants. 322 transcripts were altered in abundance in the�mia40 plants and were significantly overrepresented in theGO annotation-response to stress (biological processesfunctional categories), representing 8.4% of the changesobserved compared with 5.7% in the genome. Furthermore,analysis of the predicted location of the proteins of geneswhose transcripts changed in abundance revealed that pro-teins predicted to be located in peroxisomes were signifi-cantly overrepresented by 6.8% compared with the 1.5%observed in the genome (Fig. 5A). A MapMan overview ofdifferentially expressed genes revealed that transcriptsinvolved in responses to biotic stresses were overrepre-sented, including four superoxide dismutase genes (threecopper zinc and one manganese) (Fig. 5B). Thus, overall per-oxisomal function seemed to be affected to a greater extentthan mitochondrial function at a transcript level. Specifi-cally, transcripts from genes encoding CSD1, CSD2, and
Ccs1 were decreased in abundanceby more than 2-fold, with signifi-cant decreases also observed forCA1 (reduced �1.9-fold). Thiscorrelated with the decreasein protein observed by Westernblotting.
DISCUSSION
Characterization of the functionsof Mia40 in A. thaliana reveal con-served and novel functions com-pared with studies in yeast. Theabsence of CSD1 and Ccs1 in mito-chondria from �mia40 plantswould be predicted from studies inyeast, and thus, this appears to be aconserved function of Mia40 acrosswide phylogenetic gaps. However,the decrease in amount and activityof complex I by �40% represents anadditional role for Mia40 that hasnot been previously reported. Addi-tionally, Mia40 does not play anessential role in the import and/orassembly of small Tim proteins intoA. thaliana mitochondria, as evi-denced by the fact that the carrierimport pathway operated normally.Mia40 has also taken on additional
novel roles in A. thaliana; it is also located in peroxisomeswhere it is required for the assembly of CSD3 and affects theabundance of a protein (Aim1) associated with �-oxidation offatty acids. The changes in transcript abundance for manygenes encoding peroxisomal proteins suggest that many moreproteins are likely to be affected. Finally the absence of Mia40results in an alteration of the basal transcriptome in A. thali-ana. Global transcript analysis illustrated that for many of theproteins that decreased in abundance in Western blots, tran-script abundances also decreased, suggesting that not only isMia40 required for their assembly and stability, but that in itsabsence signals from the organelles result in these pathwaysbeing down-regulated.The functions of Mia40 in A. thaliana differ considerably
compared with those observed in yeast. Our results indicatethat it is possible for themitochondrial disulfide relay system tofunction without Mia40 in A. thaliana. This is consistent withthe findings in trypanosomes, which also lack aMia40 gene butstill successfully import small Tim proteins into the intermem-brane space (65). The different arrangement of cysteine pairs inErv1 in A. thaliana (plants) and trypanosomes (57) may allowErv1 in these organisms to function alone as a replacement oftheMia40/Erv1 system in yeast. Another possibility is that Erv1functions in another, as yet unknown, pathway or with otheruncharacterized components. Notably, the A. thaliana Erv1protein could not complement a yeast erv1 mutant, indicatingthat it functions differently (supplemental Fig. 3), as observedpreviously (55). Furthermore, the amount of Erv1 significantly
Plas d: 9.62%Mitochondria: 6.39%Peroxisome: 1.56%other cellular loca on: 82.43%
Plas d: 9.63%Mitochondria: 7.14%Peroxisome: 6.83%other cellular loca on: 76.40%
Transcripts ∆mia40 - 322 transcripts
FIGURE 5. Analysis of the changes in transcriptome of A. thaliana �mia40 plants. A, shown is the propor-tion of transcripts encoding proteins located in mitochondria, plastids, and peroxisomes in Col-0 and �mia40plants. B, shown is a MapMan visualization of changes in transcript abundance of genes encoding proteinsinvolved in stress and redox metabolism. FDR, false discovery rate; PPDE, posterior probability of differentialexpression.
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increased in A. thaliana �mia40 plants, indicating thatalthoughA. thalianaMia40may not be an essential componentfor the Erv1 disulfide relay pathway in A. thaliana mitochon-dria, it may still participate in the process. Finally, the increasein Erv1 did not compensate for all of the functions of Mia40 inmitochondria from A. thaliana as both CSD1 and Ccs1 wereabsent in �mia40 A. thaliana plants, in addition to the reduc-tion in the amount and activity of complex I.The reasons for this difference in the MIA pathway between
yeast and plants is unclear, but in plants it may relate to the factthat the intermembrane space is also the location of ascorbatebiosynthesis (26), and ascorbate can participate in oxidativeprotein folding (66). No change in total ascorbate was detectedfor �mia40 plants (supplemental Fig. 4). Thus, either the plantErv1 protein can function alone in the disulfide relay system, orif Mia40 is present, it is not essential and can be compensatedfor by an increase in the abundance of Erv1. It cannot be ruledout that other components that may be unique to the inter-membrane space in plants replace the function of Mia40. Notethat a calculation of the redox potentials of components of themitochondrial disulfide relay system suggest that shuttlingelectrons from a protein substrate (redox potential �310 to�340mV) viaMia40 (redox potential�200mV) to Erv1 (redoxpotential�320mV) presents a thermodynamically unfavorablereaction thatmay be overcome by a variety ofmechanisms (57).Thus, the absence of Mia40 in this system does not present anymechanistic barrier to the operation of a disulfide relay.Mia40 acts as a receptor for proteins imported in the inter-
membrane space in yeast (3). In A. thaliana (and by inferenceplants in general) the outermembrane receptor componentsdiffer from the yeast andmammalian systems. The plant outer-membrane Tom20 and OM64 proteins are not orthologous toyeast Tom20 and Tom70 (22, 67–69). Although A. thalianaMia40may be orthologous to yeast Mia40, it differs in function(and location). Recently, in yeast it has been shown that Mia40binds a specific nine-amino acid motif, intermembrane space-targeting signal (ITS) (70). A difference in this intermembranespace-targeting signal sequencemay represent a differentmodeof import for plant intermembrane space proteins comparedwith yeast. However, when comparing the A. thaliana inter-membrane space-targeting signal sequences to yeast, they showa high degree of similarity with at least one of the two crucialamino acids being identical across both species (70) (data notshown). This demonstrates that the binding region in the sub-strates ofMia40 is the same in yeast andA. thaliana, thus, indi-cating that this is unlikely to be the reason for the differentfunctions between yeast and A. thalianaMia40 proteins.Although there was no deleterious phenotype associated
with the deletion of Mia40, there were a number of changes inprotein abundance in mitochondria and peroxisomes and areduction in the import of some precursor proteins via the gen-eral import pathway. The abundances of Erv1 and Tim23-2were increased, but no other significant changes in componentsof the mitochondrial protein import apparatus were observed.Some components showed a slight decrease, such as Tom20-2,Tom20-3, andTom20-4 and the cytochromebc1 complex, indi-cated by the reduction in the Rieske FeS protein. However, asinactivation of two of three Tom20 protein isoforms inA. thali-
ana does not greatly affect protein import via the generalimport pathway (22), these changes in Tom20 isoforms aloneare unlikely to account for the observed changes in proteinimport.A decrease in the Nad9 subunit of Complex I of the respira-
tory chain and a parallel decrease in complex I activity wasobserved in �mia40 plants. The abundance of the other respi-ratory complexes did not differ from mitochondria from wildtype plants. Additionally, no change was observed for the alter-native oxidase at a protein or transcript level, indicating nogeneral mitochondrial stress as a result of deletion of Mia40(47). Finally,�mia40 plants did not display any of the growth ordevelopmental abnormalities associated with the absence ofcomplex I that have been previously characterized in A. thali-ana and tobacco (71, 72). As the import ability of a variety ofproteins was largely unaffected, including several complex Isubunits (CA2 and NDUFS8) (60), the reduction in the rate ofprotein import for some proteins is unlikely to be the cause forthe changes in protein abundances observed. Previously wehave characterized A. thaliana plants that have all functionalTom20 receptor components inactivated. Although import viathe general import pathwaywas decreased by 80%, therewas nodetectable affects on plant growth or abundance of complex I orother respiratory chain complexes (22). Thus, the rate of pro-tein import does not affect respiratory complex abundance inmitochondria in A. thaliana.A study analyzing the phylogenetic distribution of proteins
that are substrates for the intermembrane disulfide relay sys-tem revealed either two CX3C or two CX9C motifs with sizesbetween 9 and 18 kDa. From this study it was concluded thatthese proteins were an ancient family that are widespread invarious eukaryotic lineages (74). The functions ofmany of theseproteins (cmc1, cox17, cox19, pet 191, and som1) have beeninvestigated in yeast and are associated with cytochrome c oxi-dase assembly and to a lesser extent in the cytochrome bc1complex (12, 74). This is in contrast to what was observed herewhere cytochrome oxidasewas unaffected, and the cytochromebc1 complex was minimally affected. However, as yeast (S. cer-evisiae) lack complex I, any role for Mia40 in complex I assem-bly (or stability) would not be uncovered using this system. Ithas been reported that mutations in the human orthologue ofErv1, GFER (growth factor, augmenter of liver regeneration)results in a reduction in activity of complex I, II, and IV activity(52). However in this study, although the absence of Mia40results in an absence of Ccs1 in mitochondria, there does notappear to be any affect on assembly of cytochrome oxidase thatmight be associated with altered copper homeostasis and/orinsertion into cytochrome oxidase.The absence ofMia40 inA. thaliana also resulted in an alter-
ation of the transcriptome. The majority of the changesobserved were decreases in transcript abundance. Genesencoding peroxisomal proteins were disproportionally affectedcompared with those encoding mitochondrial or plastid pro-teins. This is consistent with the targeting and accumulation ofMia40 to peroxisomes. Several transcripts encoding peroxiso-mal proteins were down-regulated in�mia40. Two short chaindehydrogenase/reductase (SDRa and SDRb) and Kat2 areinvolved in �-oxidation (75). The peroxisomal �-oxidation
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pathway in plants metabolizes saturated long chain fatty acidsto supply energy during germination and seedling establish-ment (76). �-Oxidation not only metabolizes fatty acids butother substrates such as unsaturated fatty acids and hormoneprecursorsmay also be fed into this pathway (76, 77). It has beenproposed that short chain dehydrogenase/reductase proteins inA. thaliana play an important role in this alternate form of the�-oxidation pathway in hormone metabolism, which alsorequires Kat2 to provide the thiolase activity (75). This suggeststhat Mia40 plays some role in fatty acid metabolism in peroxi-somes, consistent with the decrease observed in Aim1 proteinabundance.Another interesting transcript, which decreased in abun-
dance in �mia40, was the homolog of the yeast Ccs1 (copperchaperone for superoxide dismutase 1 (Sod1)), AtCcs1. Mia40in yeast has been shown to be required for the biogenesis ofCcs1 and Sod1 in the intermembrane space of mitochondria(73). In A. thaliana there are three Sod1-like proteins and oneCcs1-like protein. The three Sod1 proteins are located in plas-tids (CSD2), the cytosol (CSD1), and peroxisomes (CSD3), andall three have been presumed to be dependent on the one Ccs1protein for proper function (58). Although no significantdecreases were observed in the import of Ccs1 and CSD1 intomitochondria from�mia40 plants, the lack of these proteins inmitochondria isolated from �mia40 plants indicates thatalthough Erv1 may be sufficient for import of these proteins, itcannot fulfill the role of Mia40 in assembly of Ccs1 and/orCSD1. Likewise, the absence of CSD3 in peroxisomes suggeststhatMia40 is required for the assembly of Ccs1 in peroxisomes.Western blot analysis failed to detect Ccs1 in peroxisomes inA. thaliana, even from wild type plants, suggesting it is presentin very low abundance.
Acknowledgments—We thank Prof. Harvey Millar and Dr. HolgerEubel for assistance with purification of peroxisomes.
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Mia40 Functions in Protein Assembly in Arabidopsis
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Chapter 7 The mitochondrial protein import apparatus of plants
99
Chapter 7
The mitochondrial protein import apparatus of plants
Chapter 7 The mitochondrial protein import apparatus of plants
100
Foreword to Study VI Study IV identified OM64 as a novel plant mitochondrial import component and
study V demonstrated that the function of Mia40 was different in Arabidopsis compared
with other organisms. This study aimed to determine if the components of the
mitochondrial import apparatus differed within and between plant species and non-plant
species (eg: Saccharomyces cerevisiae and Homo sapiens). This study focused on
identifying all the known mitochondrial import components in a variety of plant
species, including Chlamydomonas reinhartdii, Ectocarpusu siliculosus,
Cyanidioschyzon merolae, Physcomitrella patens, Arabidopsis thaliana, and Oryza
sativa.
The results of this study demonstrates that the core channel forming subunits of
the mitochondrial outer membrane (Tom40 and Sam50) were conserved between all
plant groups and other eukaryotes. However, the receptor components of green plants
(Chlamydomonas reinhartdii, Physcomitrella patens, Arabidopsis thaliana, and Oryza
sativa), in particular, Tom20, were not orthologous to those of yeast and humans, but
are specific to the plant lineage. It was also found that the red algae Cyanidioschyzon
merolae, contains a full Tom22 receptor subunit of the TOM complex, which is
orthologous to the Tom22 receptor of yeast. This is in contrast to the green plant
lineage, where the cytosolic receptor domain of Tom22 has been lost, suggesting that
this loss occurred after the divergence of green plants from red algae. Furthermore, the
receptors of plant mitochondrial import apparatus displayed differences between the
various plant species. Specifically, distinct motifs were present in the receptor-binding
domain of plant Metaxin, which is absent in red algae. Also, the presence of OM64 on
the outer membrane of mitochondria, was found to be only present in the higher plants
Arabidopsis and rice, suggesting it is a recent addition to mitochondrial import
components.
Findings from this study has led to the proposal that the observed functional
divergences are due to the selective pressure to sort proteins between mitochondria and
chloroplasts in plants, resulting in the differences in protein receptor components seen
between plant groups and other eukaryotes.
RESEARCH ARTICLE Open Access
An in silico analysis of the mitochondrialprotein import apparatus of plantsChris Carrie, Monika W Murcha, James Whelan*
Abstract
Background: An in silico analysis of the mitochondrial protein import apparatus from a variety of species; includingChlamydomonas reinhardtii, Chlorella variabilis, Ectocarpus siliculosus, Cyanidioschyzon merolae, Physcomitrella patens,Selaginella moellendorffii, Picea glauca, Oryza sativa and Arabidopsis thaliana was undertaken to determine ifcomponents differed within and between plant and non-plant species.
Results: The channel forming subunits of the outer membrane components Tom40 and Sam50 are conservedbetween plant groups and other eukaryotes. In contrast, the receptor component(s) in green plants, particularlyTom20, (C. reinhardtii, C. variabilis, P. patens, S. moellendorffii, P. glauca, O. sativa and A. thaliana) are specific to thislineage. Red algae contain a Tom22 receptor that is orthologous to yeast Tom22. Furthermore, plant mitochondrialreceptors display differences between various plant lineages. These are evidenced by distinctive motifs in all plantMetaxins, which are absent in red algae, and the presence of the outer membrane receptor OM64 in Angiosperms(rice and Arabidopsis), but not in lycophytes (S. moellendorffii) and gymnosperms (P. glauca). Furthermore, althoughthe intermembrane space receptor Mia40 is conserved across a wide phylogenetic range, its function differsbetween lineages. In all plant lineages, Tim17 contains a C-terminal extension, which may act as a receptorcomponent for the import of nucleic acids into plant mitochondria.
Conclusions: It is proposed that the observed functional divergences are due to the selective pressure to sortproteins between mitochondria and chloroplasts, resulting in differences in protein receptor components betweenplant groups and other organisms. Additionally, diversity of receptor components is observed within the plantkingdom. Even when receptor components are orthologous across plant and non-plant species, it appears that thefunctions of these have expanded or diverged in a lineage specific manner.
BackgroundThe endosymbiotic event giving rise to the origin ofmitochondria is thought to have occurred 1 to 2 billionyears ago [1,2]. Details of the conditions that favouredthis event and the exact identity of the host cell thatengulfed the a-proteobacterial cell are still unclear. Ithas been proposed that the endosymbiosis that gave riseto mitochondria occurred under anaerobic conditions,followed by early diversification of eukaryotic cells [3].For plastids, an endosymbiotic event occurred ~1 billionyears ago when a heterocyst forming cyanobacteriumwas engulfed [4,5]. Over time the loss and/or transfer ofgenes and genomes from the endosymbionts to the hostcell nucleus has resulted in the formation of organelles
with limited coding capacity [6-8]. The majority of pro-teins located in mitochondria and plastids are encodedby nuclear located genes, translated in the cytosol andimported into these organelles. Notably, the proteomesof both mitochondria and chloroplasts are derived froma variety of sources and are not simply a subset of theproteins derived from the ancestral endosymbiont [9]. Inthe most extreme cases, it is thought that all genes thatwere present in the endosymbiont have been lost, result-ing in specialized organelles such as hydrogenosomesand mitosomes [10].Although mitochondria have a single origin there is
variation observed between different mitochondria pre-sent in the major branches of life [10]. Mitochondria inplants contain many unique features compared to theirfungal or animal counterparts. These include a largergenome, ranging from 200 Kb to 2000 Kb in size [11],
* Correspondence: [email protected] Research Council Centre of Excellence in Plant Energy Biology,University of Western Australia, 35 Stirling, Crawley 6009, WA, Australia
Carrie et al. BMC Plant Biology 2010, 10:249http://www.biomedcentral.com/1471-2229/10/249
© 2010 Carrie et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.
extensive cis and trans splicing of introns, [12], relativelyslow rates of mutations [13,14], extensive editing ofmRNA [15] and incorporation of foreign DNA [16].Another notable feature is the presence of a branchedrespiratory chain [17]. Although fungi contain alterna-tive NAD(P)H dehydrogenases and an alternative oxi-dase, these are usually only expressed under conditionswhere the cytochrome chain is inhibited [17]. In con-trast, plant mitochondria contain components of thealternative respiratory pathways which exhibit both con-stitutive and stress induced expression [18]. Further-more, mitochondria of plants and animals havediversified in a lineage specific manner to include orexclude various biochemical pathways, such as theb-oxidation of fatty acids that occurs in peroxisomes inplants and mitochondria in animals [19].In plants, the presence of plastids in cells also adds to
the complexity of protein sorting required to avoidmis-targeting of proteins to organelles. Plastidic andmitochondrial targeting signals, referred to as transitpeptides and presequences respectively, are typicallylocated at the N-terminal end of the protein and areenriched in positively charged residues such as lysineand arginine [20]. It is not known how mis-sorting ofproteins is prevented between plastids and mitochon-dria. A combination of the predicted ability of transitpeptides and presequences to form different secondarystructures, the proposed presence of cytosolic targetingfactors and even targeting of mRNA to the surface oforganelles, may all combine to achieve the observed spe-cificity of protein targeting [21,22]. There is a mechanis-tic difference between recognition of targeting signals bypreprotein receptor proteins in plastids and mitochon-dria, the former involving a GTP/GDP cycle while noenergy requirement is observed for receptor binding inmitochondria. This mechanism among others may con-tribute to the specificity of targeting signal recognitionat the surface of each organelle [23,24].Our knowledge of the mitochondrial protein import
apparatus in plants, both experimental and predicted, islargely derived from studies in Arabidopsis, and to a les-ser extent from Solanum tuberosum (potato). Purifica-tion of the translocase of the outer membrane (TOM)complex from both Arabidopsis and potato revealedthat Tom40 and Tom7 are orthologous with those fromyeast, while Tom20 is not orthologous to yeast or mam-malian proteins [25-27]. The other import receptorscharacterized in yeast (and mammals), Tom70 andTom22, appear to be absent [28,29]. It has been shownthat plant Tom9 is the most likely equivalent to yeastTom22, but lacks the cytosolic receptor domain [30].The mitochondrial processing peptidase has been puri-fied from potato and shown to be integrated into thecytochrome bc1 complex [31,32]. This is also the case in
lower plants examined both in the elkhorn fern Platy-cerium bifurcatum and the field horsetail Equisetumarvense [33]. Biochemical purification of the prese-quence degradation peptidase (PreP) has shown that itis a dual targeted protein and that it is a zinc metallo-protease [34]. Biochemical studies have shown thatsmall intermembrane space proteins also mediate mito-chondrial carrier protein import in potato mitochondria.In addition, the plant TIM17:23 complex differs to thatin yeast in that the Tim17 in Arabidopsis contains a C-terminal extension that must be removed before it cancomplement a tim17 mutant in yeast [35,36].However, there are limited studies on the nature of
the mitochondrial protein import apparatus from otherplants, ranging from single celled algae to monocots.Thus, in order to gain a better overview of the proteinimport apparatus in plants, compared to fungal and ani-mal counterparts, an in silico analysis of these compo-nents was carried out. This was based on the fact thatcomplete genome sequences now exist for the singlecelled green algae, Chlamydomonas reinhardtii (Chloro-phyte) [37] and Chlorella variabilis, an intracellular sin-gle celled green algae photosynthetic symbiont inParamecium bursaria [38], a moss, Physcomitrellapatens (Bryophyte) [39], Selaginella moellendorffii, anancient vascular plant [40], and higher plants Oryzasativa [41], Arabidopsis thaliana [42] and Picea glauca[43] (Spermatophytes) (Figure 1). We have also includedanalysis from brown algae, Ectocarpus siliculosus(Phaeophyceae) [44] and the red algae, Cyanidioschyzonmerolae (Rhodophyta) [45] (Figure 1). Red algae repre-sent a cell lineage with a primary plastid endosymbiosisthat is proposed to have been derived from the sameevent that gave rise to the plastids in green plants, butdiverged from the green plant lineage early after thisendosymbiotic event [46]. Brown algae have obtainedtheir plastids via a secondary endosymbiosis, and con-tain four plastid envelope membranes. Thus, plastid pro-teins are first targeted to the outer membrane via ahydrophobic signal sequence and secondary targetingsignals mediate uptake into plastids [47].
Results and DiscussionTranslocase of the Outer Membrane (TOM)The TOM complex represents the gateway into mito-chondria, through which almost all mitochondrial pro-teins pass (exceptions include Fis1 [48]). It has beencharacterized from yeast, Neurospora, mammals andplants, in particular Arabidopsis. In addition to beingpurified from Arabidopsis, functional studies on theTom20 receptor components show that all three iso-forms can be deleted, resulting in a reduced rate ofimport for several precursor proteins, but no deleteriousphenotypic lesions. The complex typically contains 7
Carrie et al. BMC Plant Biology 2010, 10:249http://www.biomedcentral.com/1471-2229/10/249
Page 2 of 15
subunits, Tom70, 40, 22, 20 7, 6, and 5, but in Arabi-dopsis Tom22 is replaced by Tom9 and no orthologueto Tom70 can be identified. Arabidopsis contain a pro-tein termed OM64 that is not present in yeast or mam-mals, which appears to play a role as an importreceptor.The TOM complex fulfills the vital function of specifi-
cally recognizing mitochondrial proteins from the poolof all proteins synthesized in the cytosol. Of the sevencomponents characterized biochemically to be presentin the TOM complex from yeast, only Tom40 is con-served between yeast, mammals and plants (Figure 2,Additional file 1). While Tom40 is a b-barrel protein,there is no significant sequence similarity with bacterialb-barrel proteins [30], nonetheless, hidden Markovmodel searches define this as a universal component ofall mitochondria, including mitosomes in Entamoebahistolytica and Giardia intestinalis [49,50].Tom22 has been shown to fulfill a central receptor
role in yeast, and insertional inactivation in yeast resultsin a strong impairment of mitochondrial biogenesis,compared to the other two preprotein receptors charac-terized, Tom20 and Tom70 [51]. Searching the genomeof the red algae C. merolae for Tom22-like proteinsidentified a protein with a predicted molecular mass of20 kDa. This protein displays sequence identity and asimilar domain structure to the yeast Tom22 (Figure 2,Figure 3b). Thus, the TOM complex of C. merolaeappears to be similar to that of D. discoideum, in that itcontains a single receptor Tom22-like protein [49]. Incontrast, green plants and E. siliculosus do not contain a
Tom22 protein. Rather, they contain a Tom9 proteindomain component (Figure 2, Additional file 1). PlantTom9 is predicted to be structurally similar to yeast andmammalian Tom22, except that it lacks the cytosolicreceptor domain [30]. Thus, Tom22 has either lost thereceptor domain to form Tom9 or been replaced by adifferent protein. Irrespective of the mechanisms bywhich Tom9 arose, it appears that green plants havelost the Tom22 receptor. The presence of the Tom22receptor component in C. merolae and D. discoideumsuggests that it represented a universal mitochondrialreceptor component prior to the divergence event thatgave rise to plants verse animals and fungi.None of the receptor proteins characterized in yeast
or mammalian systems, Tom20, Tom70 and Tom22, arepresent in green plants [52,53] (Figure 2). The evolu-tionary situation for Tom22 is outlined above, andalthough a Tom20 receptor protein is present in plants,it represents a case of convergent evolution that hasbeen previously well described [27,54,55], thus, plantand yeast Tom20 proteins are not orthologous (Figure 2,Additional file 1). The third receptor component,Tom70, is only present in animals and fungi [45] (Fig-ure 1). Tom70 is not present in any green plant genome[29], a variety of searches in this study failed to detectany Tom70 like sequences in the green plant genomesinterrogated. However, in the genome of the brownalgae E. siliculosus, a protein with a similar domainstructure to Tom70 was identified (Figure 3C). ThisTom70 like protein contains an N-terminal transmem-brane domain and 11 Tetratricopeptide repeat (TPR)
Homo sapiens
Saccharomyces cerevisiae
Oryza sativa
Arabidopsis thaliana
Physcomitrella patensChlamydomonas reinhardtii
Dictyostelium discoideum
Cyanidioschyzon merolae
Ectocarpus siliculosus
Picea glaucaSelaginella moellendorffii
Chlorella variabilis
02004006008001000120014001600Million years ago
Opisthokonts
Amoebozoa
Green Plants
Red algae
Brown algae
Figure 1 Overview of the evolutionary relationship of organisms used in this study. The taxonomy database at NCBI was used to draw aphylogenetic tree, which was visualized using PHY-PI [91]. The timeline is based upon [57].
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9
Picea
Figure 2 Diversity of the TOM complex in plants. A) Schematic diagrams of the TOM complex from a selection of plant species using theTOM complex from yeast as a reference. B) A table depicting components of the TOM complex in a variety of organisms. The pink color refersto proteins that are conserved across all organisms and likely have a common ancestor. The lime green colored proteins are specific to the plantlineage. The pale green proteins are proteins that have an unknown origin. Yeast - Saccharomyces cerevisiae, Ectocarpus - Ectocarpus siliculosus(Es), Cyanidioschyzon - Cyanidioschyzon merolae (Cm), Chlorella - Chlorella variabilis (Cv), Picea - Picea glauca (Pg), Selaginella - Selaginellamoellendorffii (Sm), Chlamydomonas - Chlamydomonas reinhardtii (Cr), Physcomitrella - Physcomitrella patens (Pp), Rice - Oryza sativa (Os),Arabidopsis - Arabidopsis thaliana (At), Human - Homo sapiens, Poplar -Populus tricocarpa (Pt), Glycine - Glycine max (Gm) Zea - Zea mays (Zm).
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Figure 3 Analysis of components of the plant TOM complex. A) Phylogenetic tree of the chloroplastic Toc64 and mitochondrial OM64sequences from plants. Only sequences which showed the characteristic domain structure of Toc64, an N-terminal transmembrane domainfollowed by an amidase like domain with 3 TPR repeats at the C-terminus, were used for phylogenetic analyses. No such proteins wereidentified in Ectocarpus siliculosus, Cyanidioschyzon merolae, Chlamydomonas reinhardtii and Chlorella variabilis. For most plant species a clearToc64 and mtOM64 homologue can be identified however only 1 sequence can be identified in Physcomitrella patens, Selaginella moellendorffiiand Picea glauca which all branch closest to the Toc64 chloroplastic proteins. B) Sequence alignment of the Saccharomyces cerevisiae Tom22with the Arabidopsis thaliana Tom9 and Cyanidioschyzon merloae Tom22. While most plants contain only the Arabidopsis Tom9 like proteinCyanidioscyzon contains a full Tom22 receptor, which shows a high similarity with the yeast Tom22. C) The domain organization of the yeastTom70, Arabidopsis mtOM64 and an EsTom70 like protein. TM - transmembrane domain and the numbers correspond to the TPR repeats. At -Arabidopsis thaliana, Vv - Vitis vinifera, Gm - Glycine max, Pt - Populus tricocarpa, Os - Oryza sativa, Pp - Physcomitrella patens, Zm - Zea mays, Sc -Saccharomyces cerevisiae, Cm - Cyanidioschyzon merolae, Es - Ectocarpius siliculosus, Sm - Selaginella moellendorffii and Pg - Picea glauca.
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motifs similar to yeast Tom70 [29]. However, the levelof sequence identity is low (20%), and it is unclear ifthis protein represents a Tom70 orthologue.A protein with a predicted molecular mass of 64 kDa
(OM64) is found on the outer membrane of mitochon-dria in Arabidopsis, displaying ~70% sequence identitywith the Toc64 protein (translocase of the outer envel-ope of chloroplasts) from plastids [56] (Figure 3A). Inplant mitochondria, this protein has been shown to beinvolved in the import of some precursor proteins [53].Analysis of various plant genomes reveals that OM64appears to be present only in a sub-set of vascularplants and is absent in P. glauca and S. moellendorffii,as well as lower plant groups represented by C. rein-hardtii, C. variabilis, P. patens, E. siliculosus andC. merolae (Figure 2A). Tom7 represents an interestingcase in that it is absent in C. merolae, but present in allother plants and eukaryotes (Figure 2). TBlastx and hid-den Markov model based searches of all red algae gen-omes available failed to find this component [50]. Thuseven if it was not annotated in the genome sequence ofC. merolae these searches should detect its presence.However, it cannot be ruled out that it may have beenmissed in the sequencing and/or assembly of theC. merolae genome. Tom5 and Tom6, proteins ofapproximately 50 amino acids long, were not detectedin C. variabilis, using either plant or yeast interrogationsequences in searches. However, the small size of Tom5and 6 proteins means that it is difficult to define theirevolutionary relationship across wide phylogenetic gaps.Tom7, on the other hand appears to be orthologousacross all groups, with the exception that it cannot befound in C. merolae. Thus suggesting that the smallTOM proteins may be lineage specific, as is the case ofthe Tom20 receptor.It is evident that the TOM complex of plants displays
diversity with respect to the receptor components pre-sent. While Tom40 is universally present, the presenceof Tom20 is only evident in green plants, Tom70 is onlypresent in E. siliculosus, and OM64 appears to havearisen by a relatively recent evolutionary event as it isonly present in a variety of monocot and dicot plantsexamined and could not be detected in P. glauca andS. moellendorffii (Figure 2A and 3A). The brown algaeE. siliculosus, contains a Tom70 type receptor. As thereis no Tom70 like sequences in green plants [29], theTom70 type receptor was either derived from the speci-fic host in the symbiosis that led to the formation ofbrown algae, or alternatively, it may represent a case ofconvergent evolution, as has been observed betweengreen plants and Opisthokonts for the Tom20 receptor[27,55].An analysis of the mitochondrial protein import appa-
ratus in a variety of plants reveals that C. merolae
clearly contains a Tom22 type receptor in contrast to allother plant lineages. Thus, this component may eitherhave been lost from brown algae and green plantlineages or the presence of a Tom22 type receptor inC. merolae represents another case of convergent evolu-tion. As brown algae are proposed to have been derivedfrom red algae, after the latter branched from greenplants [57], the Tom22 receptor would have to be lostindependently in green plants and brown algae. How-ever, caution needs to be exercised, as the sequence ofE. siliculosus may not be fully representative of allbrown algae.The question of how red algae solve the sorting pro-
blem between plastids and mitochondria may relate tothe binding substrates of the receptors, that is, the tar-geting signals. Analysis of plastid targeting signals fromall plant lineages reveals that red algae (and the otherprimary plant lineage, glaucophytes) contain a phenyla-lanine residue within a few amino acids of theN-terminus, which is in a hydrophobic context [58].This ‘ancestral’ plastid targeting motif is not present inplastid targeting signals in green plants [58], and thusthe differentiation of plastid and mitochondrial targetingsignals in green plants differs to red algae. In red algae,the “phenylalanine containing” transit peptide may serveas a means for mitochondria and plastid targeting sig-nals to be recognized or rejected by plastidic or mito-chondrial receptors respectively.
Sorting and Assembly Machinery of the OuterMitochondrial Membrane (SAM)The SAM complex is required for the insertion ofb-barrel and a-helical proteins into the outer membrane[52]. The insertion of b-barrel proteins into the mito-chondrial outer membrane is conserved from bacteria tomitochondria and plastids, where Omp85, Sam50 andToc75 are orthologous b-barrel proteins that are essen-tial for this process [59,60]. However, apart from thiscentral component, there are no other conserved com-ponents identified for the insertion of b-barrel proteinsinto membranes from bacteria to mitochondria andplastids (Figure 4A and 4B). In yeast, four additionalcomponents are involved; Sam35, Sam37, Mdm10 andMim1, with Sam35 representing an essential component.As the SAM complex has not been biochemically char-acterised from mammalian or plant systems, any addi-tional components are unknown in these systems. Thegenome of D. discoideum has a gene encoding Sam50,but lacks the other components identified in yeast. AsD. discoideum is an amoeba that diverged from Opistho-konts after this lineage had split from plants, this sug-gests that the additional components observed in yeastarose after the lineage divergence of plants from othergroups. Although additional components are likely to be
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B)
C) Yeast Sam37TM GST1 GST2
Arabidopsis MetaxinM1 M2
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Human Metaxin2TMGST1 GST2
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Figure 4 The SAM complex of plants. A) Schematic representation of the SAM complexes found in yeast and plants. B) Table indicating thepresence or absence of a component of the SAM complexes found in plants. C) Representation of the different domains of yeast Sam37,Human Metaxin and Arabidopsis metaxin proteins. All three share similar Glutathione S-transferase (GST) domains. However the location of thetransmembrane domains (TM) differs. A motif analysis search of plant metaxin sequences identified Motif 1 to regions between amino acids 37and 79 and motif 2 between amino acids 104 and 155 in Arabidopsis. These motifs appear only in plant like Metaxins and near a regionrequired for protein binding. Colors and abbreviations are the same as Figure 2.
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present in the SAM complexes from plants, they areunlikely to be orthologous to the components in yeast.In Arabidopsis, a protein called Metaxin has been
shown to be involved in the import of b-barrel proteinsinto the outer membrane. The deletion of Metaxin isnot lethal in Arabidopsis, although plants are sterile andgrow poorly [53]. Deletion of Metaxin results in a largeup-regulation of transcript abundance for genes encod-ing the mitochondrial b-barrel proteins porin andTom40, with an accumulation of porin observed in thecytosol, indicating that Metaxin plays a role in the inser-tion of b-barrel proteins in plants. Mammalian genomescontain two genes encoding Metaxin, in fact the plantMetaxin protein was identified using blast searches ofthe mammalian Metaxin protein [53] (Figure 4C). Mam-malian Metaxin has also been shown to be involved inthe import of b-barrel proteins into the outer membraneof mitochondria [61]. Mammalian Metaxin does identifywith Sam37 in a blast search, although the sequenceidentity is very low (Additional file 1). A number of fea-tures distinguish plant and animal Metaxins fromSam37 in yeast. Firstly, human and Arabidopsis Metax-ins are anchored to the outer membrane in the oppositeorientation compared to Sam37 (Figure 4C). YeastSam37 is anchored to the mitochondrial outer mem-brane by an N-terminal transmembrane domain,whereas human and Arabidopsis Metaxins containC-terminal transmembrane domains. Secondly, humanand Arabidopsis Metaxins contain conserved glutathioneS-transferase (GST) domains (Figure 4C). Plant and ani-mal Metaxins are distinguished by the fact that Metaxinis not found in a complex with human Sam50 [61],whereas plant Metaxin is in a complex with plantSam50 (Duncan and Whelan - unpublished data). InArabidopsis Metaxin there are two conserved motifs ina region critical for binding that are only found in planttype Metaxins (Figure 4C motif 1 and 2). It is also ofinterest to note that while Trypanosomes do contain aMetaxin like protein [62], there are no Metaxin orSam37 like proteins identified in D. discoideum [49] orC. merolae in this study. The presence of a Metaxinprotein in E. siliculosus may be derived from the hostcell. Thus, plant and animal Metaxins may be ortholo-gous, but functions are likely to have diverged overtime. Biochemical characterization of the plant SAMcomplex would provide information on the accessoryproteins of this complex and provide a clearer picture ofthe evolutionary nature of the accessory subunits in thiscomplex.
Intermembrane space - Mitochondrial intermembranespace import and Assembly (MIA) and Tiny TIMsThe intermembrane space contains two sets of proteinsthat are essential for cell viability in yeast. The tiny TIM
proteins 8, 9, 10 and 13 appear to be present in a widevariety of eukaryotes (Figure 5A). They play an essentialrole in the import of carrier proteins into the innermembrane and also the assembly of b-barrel proteinsinto the outer membrane [52]. It has been proposedthat they arose from an ancestral protein present in theoriginal host that housed the mitochondrial endosym-biont [63]. There are eukaryotes that lack the smallTims (Trichomonas vaginalis and Encephalitozoon cuni-culi) or only contain one small Tim protein (Cryptospor-idium hominis) [64,65], indicating that they are notabsolutely essential, even though these organisms con-tain carrier type proteins on the inner membrane thatshould require these components for import. Thus, thelack of small Tims is likely to be a derived situationassociated with the presence of highly modified mito-chondria (i.e. mitosomes) in these organisms.The MIA pathway is the most recently described
import pathway for mitochondrial proteins. It consistsof two essential proteins in yeast, Mia40 and Erv1,which catalyse the oxidative folding of proteins whenthey enter the intermembrane space. Substrates of thispathway are proteins that contain conserved cysteineresidues that undergo oxidative protein folding in theintermembrane space. Both Mia40 and Erv1 are essen-tial proteins in yeast, with Mia40 proposed to act as theintermembrane space receptor for proteins [66]. Whilstdetailed structural and mechanistic analysis has beencarried out on this system in yeast [67], little is knownabout the components in other organisms. Interestinglythe apparent lack of a gene encoding Mia40 in trypano-somes suggests that this pathway may display variationsbetween species [68].For the MIA machinery, orthologues of Mia40 and
Erv1 are present in yeast, humans and plants (Figure5A). Although Hot13 has been reported to be wide-spread in eukaryotes, analysis of the proteins identifiedindicates it is ~600 amino acids long and most likely atranscription factor in green plants. Brown algae containa protein similar to yeast Hot13 (Figure 5A, Additionalfile 1). However, given the small size of this proteinwith conserved metal domains it is unclear if it is ortho-logous to the yeast protein. Although plant Erv1 andMia40 are orthologous to their yeast counterparts, theprimary structure of the protein differs (Figure 5B), sug-gesting possible mechanistic differences. Deletion ofMia40 in Arabidopsis is not lethal, and in fact normalgrowth and development are observed [69]. Erv1 isessential in Arabidopsis and analysis of the primarysequence indicates that the arrangement of cysteines dif-fers to that in yeast (and humans). Arabidopsis Erv1 issimilar to that found in Trypanosoma brucei and a pro-tein called Erv2 that is located in the endoplasmic reti-culum of yeast (which operates without a Mia40 like
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A)
B) Yeast Erv2p (196 AA)
Yeast Erv1p (189 AA)
Human Erv1 (205 AA)
Arabidopsis Erv1 (191 AA)
Trypanosome Erv1 (273 AA)
CXXC C-16-CFAD
CGC
CXXC C-16-CFAD
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CXXC C-16-CFAD
CXXXXC
E. cuniculi (162 AA)
CXXC C-16-CFAD
CXC
Mia40 essential
Mia40 non essential
No known Mia40
CXXC C-16-CFAD
Figure 5 Components of the MIA and IMS protein import apparatus of plants. A) A table displaying the components of the small Timproteins and MIA pathway components of plants. B) Schematic diagram of the different Erv1 sequences found in different organisms. The greyregion represents the most conserved region between different Erv1 sequences containing the redox centre, the FAD binding site and twoconserved cysteine domains (CXXC and C-16-C). The location of the third cysteine pair differs between organisms which seems to be dependenton either a Mia40 protein being present or whether that Mia40 is essential or not. Abbreviations are the same as Figures 2 and 4.
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protein) (Figure 5B). Given that the import of the smallTim proteins and carrier proteins is normal in Arabi-dopsis plants that lack Mia40 [69], mechanistically Erv1can function without Mia40 in oxidative protein foldingin the intermembrane space. Analysis of the genome ofD. discoideum indicates that Mia40 is present [68]. AsMia40 is absent in Trypanosoma brucei, Encephalitozooncuniculi [65,68], and the brown algae E. siliculosus(Figure 5A, Additional file 1), this suggests that the pre-sence of Mia40 in plants is a primitive situation, but itsfunction(s) differ in various lineages.
Translocases of the inner membrane (TIMS)The inner mitochondrial membrane contains two trans-locases, the TIM17:23 complex that is responsible forthe import of proteins via the general import pathway,and the TIM22 complex, that is responsible for theimport of carrier proteins into the inner membrane. TheTIM17:23 complex is responsible for the import of pro-teins that contain N-terminal targeting signals into oracross the inner mitochondrial membrane [23]. Thiscomplex contains 9 components in yeast and several ofthe components are essential in yeast [52]. Tim23 formsa presequence and voltage sensitive channel [70], whileTim17 plays a crucial role in voltage sensing [71,72].The TIM17:23 complex can be divided into the PAMcomplex, the presequence assisted motor consisting offive subunits (Tim44, HSP70, Pam 16, 17 and 18) andthe membrane components of Tim17, 23, 21 and 50.The TIM22 translocase is responsible for the import ofproteins that contain internal targeting signals and con-tain multiple (4 or 6) transmembrane spanning regionsinto the inner membrane [23]. In contrast to theTIM17:23 complex, the mechanistic details of how itoperates are not yet fully understood. However Tim22has been shown to have channel activity that is onlyactive in the presence of a substrate protein [73]. Inyeast it contains three accessory proteins, Tim54, 18and 12 [52]. However no details on the composition ofthis complex in other organisms have been reported.In contrast to the TOM complex on the outer mem-
brane, eight of the nine components of the Tim17:23complex are conserved between yeast, humans andplants (the only difference being that yeast contain aPam17 protein not present in humans and plants)(Figure 6A, Additional file 1). It has been previouslyproposed that the channel forming subunits of this com-plex, Tim23 and Tim17, are derived from amino acidtransporters in bacteria, specifically LivH, and defined afamily of proteins termed PReprotein and Amino acidTransporters (PRAT) [74]. It seems that originally therewas one PRAT type protein that subsequently divergedto give rise to the three different PRAT proteins typi-cally found in mitochondria [75]. In some organisms a
single PRAT protein exists, which is likely a derivedcondition where the other PRAT proteins have beenlost [65]. Additionally, not all subunits of the TIM17:23complex are observed in all organisms, i.e. the absenceof Tim50 in D. discoideum [49], suggests that accessorysubunits can be lost.In the case of the TIM22 complex, the translocase
responsible for the import of metabolite carriers, ormultiple spanning proteins of the inner membrane,including the PRAT proteins themselves, only theTim22 component is conserved. In fact Tim18 andTim54, along with Tim12, are only found in yeast andnot in other organisms including plants. Thus, the addi-tional components of this translocase are yet to be char-acterized in other organisms.Although the TIMs seem to be better conserved in
terms of orthology compared to the TOM complex,there are notable differences in plants. Firstly, the familyof PRAT proteins has greatly expanded in plants com-pared to yeast and mammals. In Arabidopsis there are17 members, rice has greater than 24 members andexamination of C. reinhardtii and P. patens reveal 5 and21 members respectively. Some of these PRAT proteinsare located in plastids, while others are found in mito-chondria [76]. In addition to the greater number ofPRAT proteins in plant genomes, Tim17 in plants variesin size from 133 amino acids to 252 amino acids, thedifference to yeast Tim17 of 158 amino acids is at theC-terminal end of the protein (Additional file 1).A C-terminal extension is found on Tim17 proteinsfrom C. merolae through to Arabidopsis. It has beenshown in Arabidopsis that this C-terminal extension isexposed on the outer surface of the outer membrane[36] and Arabidopsis Tim17 can only complement ayeast Tim17 mutant if this extension is removed [36].In order to investigate possible function(s) of the
Tim17 C-terminal extension in plants we conducted amotif search on all the identified Tim17 extensions.A distinct motif was detected (Figure 6C). Using thismotif in blast searches identifies a number of differentnucleic acid binding proteins (Additional file 2). Thissuggests that Tim17 in plants may be able to bind RNAand/or DNA. As plant mitochondria import tRNAs andhave recently been shown to bind mRNA [22,77], sug-gests a possible role for Tim17 in the binding and/orimport of nucleic acids into plant mitochondria.
Mitochondrial processing peptidase(s)Mitochondria require a number of peptidases to removethe targeting signals from proteins before or after theyare assembled into functional protein complexes. Thesepeptidases range from activities that remove the target-ing signals, such as mitochondrial processing peptidase(MPP) and intermediate processing peptidase (IMP)
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A)
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AtTim17-2 243 Amino acidsM1TM TM TM TM
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Figure 6 The TIM17:23 and TIM22 complexes of plants. A) A schematic representation of the TIM17:23 complex from yeast and plants.B) A table displaying the components of the TIM17:23 and TIM22 complexes in all plant species analysed. C) A diagram showing ArabidopsisTim17-2 that contains four transmembrane domains (TM) and an extra C-terminal extension. This C-terminal extension is found in all higherplant species and motif analysis of the C-terminal extension identified to highly conserved regions of which both are related to nucleic acidbinding proteins. Colors and abbreviations are the same as Figures 2 and 4.
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[23], to the removal of a single amino acid from pro-teins that have already had the targeting signal removed[78], to the presequence degrading peptidase (PreP) thatdegrades the targeting signals once they have beenremoved [34].Plant mitochondria contain a number of orthologous
proteins in comparison with the various processing pep-tidases of yeast mitochondria (Additional file 1). At asequence level the processing peptidases found in plantmitochondria look similar to those of yeast, however,functional investigations have revealed a number of dif-ferences. One major difference between yeast and plantsis the location of the mitochondrial processing peptidase(MPP). In yeast both a and b MPP subunits are locatedin the matrix, however, in plants it has been demon-strated that they are integrated into the cytochrome bc1complex located in the inner membrane [31,32]. Addi-tionally, the presequence degrading peptidase of yeast islocated in the intermembrane space whereas in plants itis located in the matrix [79,80]. Interestingly, the prese-quence degrading peptidase of plant mitochondria isalso dual targeted to plastids where it degrades plastidtargeting signals [79].In terms of the processing site recognition by MPP in
plants, it has been reported that the majority of plantmitochondrial presequences fall into 2 classes. In class 1the processing signal is a -2 Arg residue while in class 2presequences the signal is a -3 Arg residue [20,81], simi-lar to what has been reported for yeast and mammals[23]. However, it has been demonstrated that in factthere is a second processing step in yeast [82], a novelpeptidase called intermediate cleavage peptidase (Icp55)was found to process mitochondrial presequences afterMPP, cleaving only 1 amino acid from the N-terminus,turning the proposed -2 cleavage signal into a -3 clea-vage signal [78]. It is tempting to speculate that asplants contain an orthologue of Icp55 that the samecleavage is occurring, however this awaits experimentalconfirmation. Despite the orthology between many plantpeptidases and those in other organisms it is still neces-sary to define their specific functions in plants. It hasbeen demonstrated that the plant orthologue of therhomboid protease from yeast does not carry out thesame processing roles/activities in plants such as Arabi-dopsis [83].
ConclusionsThe plant mitochondrial import apparatus displaysmany differences compared to other non-plant organ-isms and between plant groups. The TOM complex inplants displays the most variability in that as many asfive different TOM complexes exist in plants when redalgae, brown algae and green plants are considered.Even in the green plant lineage variation is observed
with OM64 only being present in monocot and dicotplants. While the composition of the other protein com-plexes may appear more conserved, the lack of biochem-ical characterization of these complexes in any plantgroup means that the presence of plant specific acces-sory subunits in various lineages cannot be judged.Additionally for some proteins, such as Mia40 andTim17, functions have expanded in plants compared tothose characterized in yeast.
MethodsThe protein sequences for all of the known mitochon-drial protein import components from Saccharomycescerevisiae (Tom20, Tom70, Tom71, Tom40, Tom22,Tom5, Tom6, Tom7, Sam50, Sam37, Sam35, Mdm10,Mim1, Mia40, Erv1, Hot13, Tim9, Tim10, Tim8, Tim13,Tim12, Tim22, Tim54, Tim18, Tim23, Tim17, Tim50,Tim21, mtHsp70, Mge1, Tim44, Pam18, Mdj2, Pam16,Pam17, MPPa, MPPb, Oct1, Imp1, Imp2, Som1, Yta12,Yta10, Yme1, Mgr1, Mgr3, Pcp1, Icp55, Oxa1, Mba1,Cox18, Pnt1, Mss2, Mdj1, Hsp60, Hsp10, Hsp78 andZim17) were downloaded from the NCBI protein data-base (http://www.ncbi.nlm.nih.gov/protein/). TheMetaxin protein sequences were also obtained fromHomo sapiens. Using the above protein sequences Blastp[84] searches of the protein sequences from Physcomi-trella patens, Selaginella moellendorffii, Chlamydomonasreinhardtii, Arabidopsis thaliana, Oryza sativa, Zeamays, Vitis vinifera, Glycine max and Populus tricocarpawere performed using the Phytozome (http://www.phyto-zome.net) database. Blastp [84] searches of Cyanidioschy-zon merolae were performed using the Cyanidioschyzonmerolae genome project website (http://merolae.biol.s.u-tokyo.ac.jp/). Blastp [84] searches of Ectocarpus siliculo-sus were performed at the Bioinformatics online genomeannotation system website (http://bioinformatics.psb.ugent.be/webtools/bogas/overview/Ectsi). Blastp [84]searches of Chlorella variabilis NC64A genome [38] wasperformed at the Chlorella genome website (http://gen-ome.jgi-psf.org/ChlNC64A_1/ChlNC64A_1.home.html).To identify mitochondrial import components of Piceaglauca tblastn [84] searches were carried on ESTsequences [43] at the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi).All multiple sequence alignments were carried out
using MAFFT [85] and visualized using Multiple alignshow (http://www.bioinformatics.org/sms/multi_align.html). The program IQPNNI [86] was used to recon-struct a maximum likelihood phylogeny assuming theWhelan and Goldman model [87]. Phylogenetic treeswere finally visualized using the program Geneious(http://www.geneious.com).TMpred (http://www.ch.embnet.org/software/TMPRED_
form.html), TMHMM (http://www.cbs.dtu.dk/services/
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TMHMM/), and DAS (http://www.sbc.su.se/~miklos/DAS/) [88] were used in the prediction of transmem-brane regions. TPR repeats were predicted usingTPRpred (http://toolkit.tuebingen.mpg.de/tprpred) [89].Motif analysis was performed using MEME (http://meme.nbcr.net/meme4_4_0/cgi-bin/meme.cgi) usingdefault parameters for all plant like Metaxin sequencesand sequences of the plant Tim17 extensions [90].
Additional material
Additional file 1: Supplementary table 1. The mitochondrial importmachinery of plants.
Additional file 2: Supplementary table 2. The top 50 proteinsidentified using the conserved motifs on the C-terminal of plantTim17 in a Blastp search.
AbbreviationsErv1: Essential for respiration and vegetative growth 1; FAD: Flavin adeninedinucleotide; GDP: Guanosine diphosphate; GTP: Guanosine diphosphate;GST: Glutathione S-transferase; Hot13: Helper of Tim protein 13; Icp55:Intermediate cleavage peptidase of 55 kDa; Mdm10: Mitochondriadistribution and morphology protein 10; MIA: Mitochondrial import andassembly; Mim1: Mitochondrial import 1; MPP: Mitochondrial processingpeptidase; OM64: Mitochondrial outer membrane protein of 64 kDa; Omp85:Outer membrane protein of 85 kDa; PRAT: Preprotein and amino acidtransporter; TIM: Translocase of the inner membrane; TOC: Translocase of theouter envelope of chloroplasts; TOM: Translocase of the outer membrane;TPR: Tetratricopeptide repeat; SAM: Sorting and assembly machinery.
AcknowledgementsThis work was supported by an Australian Research Council Centre ofExcellence Grant CEO561495.
Authors’ contributionsCC carried out the data analysis with the help of MM. JW oversaw theanalysis, design and implementation. CC, MM and JW drafted themanuscript. All authors read and approved final manuscript.
Received: 26 August 2010 Accepted: 16 November 2010Published: 16 November 2010
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Chapter 8 General discussion
116
Chapter 8
General discussion
Chapter 8 General discussion
117
General discussion Dual targeting in plants was first uncovered in 1995 and now more than
50 proteins are known to be dual targeted to mitochondria and plastids in Arabidopsis
(Creissen et al., 1995; Carrie et al., 2009). While the vast majority of plant dual targeted
proteins are targeted to mitochondria and chloroplasts, an increasing number are being
discovered in other organelles; chloroplasts and nucleus (Schwacke et al., 2007),
chloroplasts and peroxisomes (Reumann et al., 2007; Sapir-Mir et al., 2008),
chloroplasts and endoplasmic reticulum (Levitan et al., 2005), chloroplasts and cytosol
(Thatcher et al., 2007), and mitochondria and peroxisomes (Studies II and III). The
functions of dual targeted proteins in plants also vary greatly; from tRNA synthetases
(Duchene et al., 2005), DNA polymerases (Christensen et al., 2005), peptidases
(Bhushan et al., 2003) and antioxidant defence proteins (Chew et al., 2003). However
the mechanisms and reasons for dual targeting are yet to be fully understood.
7.1 Defining dual targeted proteins: targeting vs accumulation studies
Our understanding of the reasons and mechanisms of protein dual targeting has
been limited by the relatively slow pace at which dual targeted proteins have been
characterised. The main reason for the slow pace at which dual targeted proteins have
been identified, is that demonstrating that a protein is dual targeted is more difficult than
defining a single location for a protein. Consequently, most dual targeted proteins have
been uncovered in small or single protein studies. One exception is a study by (Duchene
et al., 2005), which characterised a large number of Arabidopsis tRNA synthetases as
dual targeted proteins. In fact, several proteins defined as dual-targeted proteins in this
thesis had been previously defined as targeted to a single location (Study II and III).
Thus the burden to identify dual targeting is greater than compared to single location
proteins.
The location of a protein in a cell can be assessed in a number of ways,
including computational predictions, targeting uptake assays, and accumulation studies
(Millar et al., 2009). Each approach has strengths and weaknesses, and to accurately
determine if a protein is located in one or more locations, the use of multiple lines of
evidence is necessary. Several prediction programs are now available to predict a
proteins targeting ability (Claros and Vincens, 1996; Hua and Sun, 2001; Bannai et al.,
2002; Emanuelsson et al., 2003; Guda et al., 2004; Small et al., 2004; Nair and Rost,
2005; Hoglund et al., 2006; Emanuelsson et al., 2007; Horton et al., 2007). These
Chapter 8 General discussion
118
programs yield varying results depending on the type of algorithms used (weight
matrix, neural networks, hidden markov models, and support vector machines), the set
of protein sequences used in training the program, the range of locations predicted, the
predicted hydrophobicity of the input sequence, and the significance level used as a cut-
off (Emanuelsson et al., 2007). In terms of dual targeted proteins, the current batch of
programs are only a guide at best, especially for proteins targeted to both mitochondria
and chloroplasts. Due to the similarity of the targeting signals between these two
organelles and given that most programs are not trained with dual targeted proteins,
predictions on dual targeted proteins have varying levels of accuracy. However, in
recent years, there have been several attempts to predict dual targeted proteins in plants
(Schwacke et al., 2007; Mitschke et al., 2009). The accuracy of these predictions are
still quite limited, and more experimental evidence is required to accurately assess the
quality of these predictions. Never the less, predictions, do offer a number of
advantages, including; speed and ease of use, unbiased by the user, and predictions form
a platform to establish new lines of experimental inquiry. For example, three NAD(P)H
dehydrogenases that were previously experimentally defined as mitochondrial
(Michalecka et al., 2003), were predicted to also be peroxisomal, and subsequent
experimental testing confirmed them to be dual targeted to both mitochondria and
peroxisomes (Study III). Thus, predictions can be a valuable tool contributing to the
identification of dual targeted proteins.
Most experimental testing of protein targeting is conducted using either in vitro
or in vivo targeting uptake assays. In vitro import assays are generally performed by
purifying the organelle of interest, and testing whether the protein of interest enters the
isolated organelle in solution. Import is then assessed by observing if the incorporated
protein is protected from an externally added protease. In vitro import assays typically
have a low false positive rate as generally only the proteins found in an organelle are
imported in vitro. However, in vitro import assays are only performed with one
organelle, which is vastly different from the actual cellular landscape that contains
several organelles. Thus using this technique it may never be known whether a protein
is targeted to multiple organelles. A good example of this is NDC1, which was
previously only imported into mitochondria and subsequently assigned as having a
solely mitochondrial location (Michalecka et al., 2003). However, work in study III
revealed that it could also be imported and targeted to chloroplasts. Therefore, while in
vitro import assays work well for single organelle localised proteins, they fail to
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reproduce the complex intracellular environment and thus cannot reveal the targeting
preference between multiple organelles.
The use of GFP and its variants as passenger proteins for in vivo targeting
studies has become routine in recent years. The main advantage of in vivo targeting
assays is that the cellular environment is maintained, and thus protein uptake into all
organelles is tested simultaneously. However, the primary source of error in in vivo
targeting experiments relates to the position of the passenger protein (e.g. GFP). Placing
the passenger protein at the wrong location or using only part of the protein of interest
are common mistakes in targeting assays (Millar et al., 2006). It is common practice
when determining protein targeting, to attach only the N-terminal (50 – 100 amino
acids) to the passenger protein. Notably, this ignores targeting signals at the C-terminus
and throughout the protein. The mitochondrial NAD(P)H dehydrogenases are good
examples of this practice, as previous targeting studies using the N-terminus assigned a
mitochondrial only location (Michalecka et al., 2003). The peroxisomal targeting
signals at the C-terminus of the these three proteins were missed, and thus this
technique failed to identify the peroxisomal location (Study III). Also, when the full
protein sequence of NDC1 was fused to GFP, it was shown to be dual targeted to both
chloroplasts and mitochondria (Study III). There are now several studies showing that
for dual targeted proteins, the nature of the passenger protein is important, and that
targeting signals that support dual targeting of one passenger protein do not support the
dual targeting of alternative passenger proteins (Chew et al., 2003).
As with in vitro import assays, there are also important considerations required
for in vivo targeting studies. The first relates to the translation product(s) used in the
targeting assay. It has become apparent that alternative splicing can produce proteins
with different targeting signals, e.g. AP2 (Study II), isoaspartylmethyltransferase 2
(Dinkins et al., 2008) and glutathione S-transferase F8 (Thatcher et al., 2007).
Therefore, where possible, it is imperative to analyse all possible alternative transcripts
or gene models for a protein, in order to assess possible targeting to different locations.
The second consideration in targeting studies is the type of tissue used for the assay. In
study II, it was demonstrated that the amount of dual targeting of a protein was different
in different tissue types. In some cases, the GFP labelling of mitochondria and
chloroplasts were equal, but for other proteins it was seen that the chloroplast signal
dominated the mitochondria signal in Arabidopsis cell culture. However, when the same
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protein was analysed in onion epidermal cells, the intensity of fluorescence changed
back to be almost equal between organelles (Study II). This is consistent with a previous
study, which reported that the dual targeting of the protein sigma factor 2B was only
observed in one cell type (Beardslee et al., 2002). It has also been reported that GFP
fluorescence intensity differs between replicate experiments performed on dual targeted
proteins (Pujol et al., 2007). Thus, attention to the details of what is tested and where,
should be considered in the design and interpretion of in vivo targeting studies.
The ability of a protein to target a specific location does not necessarily lead to
accumulation within that location. It has been evidenced that a number of proteins are
first targeted to one location (e.g. endoplasmic reticulum) before being transported to a
second and final organelle (Tabak et al., 2003; Villarejo et al., 2005). The use of
chimeric constructs in targeting studies may therefore mask secondary sorting signals
and thus incorrectly determine location. Immunoblotting, immunolocalization and
activity assays are common approaches used to define the location or accumulation of
proteins. These assays are only informative when the assay is specific, which is
generally not the case in activity based assays, and varies with different antibodies as
specificity of antibodies can be difficult to demonstrate. Hence this lack of specificity
has led to the use of peptide mass spectrometry approaches to define the location of
large numbers of proteins.
Subcellular proteomics involves the cataloguing of components of an isolated
organelle after cell disruption. Subcellular proteomics has facilitated the designation of
organelles for over 4600 proteins in Arabidopsis (Heazlewood et al., 2007). However,
proteomics is still largely based on the purification of organelles from plant tissues, and
with the increasing sensitivity of new mass spectrometers, there is increasing
identification of low abundance contaminants. This has been somewhat overcome by
the use of quantitative comparison of organelle protein fractions with different degrees
of enrichment for the specific organelles of interest (Eubel et al., 2008; Huang et al.,
2009). However, many proteins are still described in protein databases as being found in
more than one subcellular location (Heazlewood et al., 2007). These proteins location is
then left unresolved until it is identified as targeted to a specific organelle or found to be
a contamination of an organelle fraction. Kat2 is a good example of a protein defined as
mitochondrial by proteomics but subsequently found to only target peroxisomes in
Arabidopsis (Study I). It has to be noted that subcellular proteomics is also limited, in
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that certain classes of proteins are rarely detected (e.g. very small, hydrophobic and/or
basic). Another limitation of proteomics is eclipsed distribution, a phenomenon
whereby a protein that is targeted to two locations, is only identified in one, due to the
uneven distribution of the proteins abundance between the two organelles (Regev-
Rudzki and Pines, 2007).
A good example of a protein with an eclipsed distribution is the yeast
aconitase 1 protein, of which 95% is targeted to mitochondria and the remaining 5% is
located within the cytosol (Regev-Rudzki et al., 2005). The challenge in identifying
proteins with eclipsed distribution is the presence of artifacts derived from organelle
leakage or fraction contamination, scenarios commonly present in traditional methods,
such as immunoblots or activity assays. The use of in vivo tagging assays are also
limited, as low abundance proteins may not be detected, as the associated level of
fluorescence may be to low or the passenger protein may affect targeting to the second
location. Hence there is a need for specific assays to determine if a protein shows an
eclipsed distribution (Regev-Rudzki and Pines, 2007). One such assay makes use of
split reporter genes such as GFP (Ozawa et al., 2003; Ozawa et al., 2005) or β-
galactosidase (Karniely et al., 2006). The advantage of this method is that the large
amount of protein in one location does not produce a signal on its own and subsequently
does not mask the signal from the eclipsed location. A second method relies more on
genetics and involves knocking out the gene of interest and replacing it with the protein
only targeted to one of its locations (Regev-Rudzki and Pines, 2007). This not only
demonstrates that the protein is dual targeted, but also permits functional analysis.
In order to reduce the error rate in defining proteins as dual targeted or location
specific, a combination of targeting and accumulation assays are recommended. In
studies focused on small numbers of proteins, a targeting assay such as a fluorescent
tagging should be combined with an accumulation assay on the protein to independently
validate localisation and give complimentary biologically relevant information. In other
words, the experiment must be designed in a manner that allows accurate determination
of dual targeting and relative abundance. With large scale approaches, such as sub-
cellular proteomics, it would be beneficial to know the error rate of defining specific
locations. Often, lists of proteins in publications may have known contaminants
removed, thus full access to the raw data is important. With large scale expression
studies, it is mandatory to deposit raw microarray data files in publicly available
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databases. This is also necessary for proteomic studies, so that other researchers can
carry out independent analysis. The availability of raw datasets and their independent
analyses will allow localisation errors to be determined and rectified.
7.2 Mechanisms of dual targeting – signals and import receptors
The signals that define a protein as dual targeted have been intensively studied
(Chew et al., 2003; Berglund et al., 2009). There are two different types of signals that
define a protein as dual targeted. Proteins can be dual targeted due to multiple
translation start sites, thus producing two different proteins from a single transcript.
Alternatively, a single protein is produced from a mRNA that is targeted to two or more
organelles. In the instance were a gene produces two (or more) translation products,
either by multiple RNAs or multiple translation start sites, the proteins produced contain
separate targeting signals for each organelle. A protein from a single translation product
can be dual targeted due to an ambiguous targeting signal or two separate targeting
signals in the same protein, or even reverse translocation. In plants, the most studied
dual targeted proteins appear to have ambiguous targeting signals and tend to be
proteins targeted to both mitochondria and chloroplasts. In this case, the ambiguous
targeting signal displays most of the properties of both mitochondrial and chloroplastic
targeting signals; enriched in hydroxylated, hydrophobic, and positively charged amino
acids, and a low abundance of negatively charged residues. Analysis of a variety of
ambiguous targeting signals has shown that deletions in the signal affects targeting to
both organelles, while smaller changes, such as single amino acid substitutions, can
affect targeting to one organelle more than another (Rudhe et al., 2002; Chew et al.,
2003; Berglund et al., 2009). Overall, it has been concluded that the dual targeting
signal is distributed throughout the targeting peptide and, importantly, it cannot be
deduced what defines a signal as a dual targeting signal, compared to a plastid or
mitochondrial specific signal.
Using two distinct signals, several proteins have now been discovered to target
to both the mitochondria and peroxisomes (Study II and III), along with chloroplasts
and peroxisomes (Reumann et al., 2007). This dual targeting is achieved either by an
N-terminal mitochondrial or chloroplastic targeting signal and a C-terminal peroxisomal
targeting signal (Study II and III)(Reumann et al., 2007). This mechanism of dual
targeting raises the question of which signal is recognised, and if ‘competition’ for the
protein occurs between the organelles. Although these questions are unanswered, they
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raise more questions about the levels of regulation of gene expression, as now
regulation of the targeting between organelles can be an important point of regulation.
One point of regulation has been speculated to involve the import pathways used for the
import of dual targeted proteins into their respective organelles.
Little is known about the role of organelle import receptors in the import of dual
targeted proteins. The dual targeted proteins identified to date are thought to be required
in a number of different plastid types, which suggests they might employ different
receptors to the Toc64 and Toc159 pathways used for photosynthetic proteins (Soll and
Schleiff, 2004). It has also been speculated that because there are four members of the
Toc159 family (Toc159, Toc132, Toc120, and Toc90), one of them may be specialised
in the import of dual targeted proteins (Jarvis, 2008). However, no experimental studies
to date have determined the import pathways or receptors for dual targeted proteins in
plastids. It has been recently demonstrated that the different A-domains of Toc159
receptors regulate their selectivity for precursor binding thus it is possible that this
differential selection may also affect the import of dual targeted proteins (Inoue et al.,
2010).
In the case of mitochondrial receptors involved in the import of dual targeted
proteins, there is some functional/experimental data on the nature of the receptor
proteins involved. In a double knockout of Tom20-2/-3, leaving only one functional
Tom20-4 isoform, it was observed that the import of GR was higher when compared to
wild-type, even though the import of several other proteins was decreased (Study IV).
In the Tom20 triple knockout, the import of GR remained at the same level compared to
wild-type, even though all other precursors were significantly decreased in import
(Study IV). These findings led to the hypothesis that GR and possibly other dual
targeted proteins, might utilize an alternative import receptor, when compared to several
mitochondrial specific proteins. One such hypothesised receptor was OM64, because it
showed a high similarity to the Toc64 receptor from chloroplasts. However, OM64 was
determined to have no specific role in the import of GR and other dual targeted proteins
(Study IV). The only receptor like protein to have a major effect of the import of GR
was Metaxin. In knockouts of Metaxin, GR import was significantly reduced, and in a
competitor import reaction with in vitro synthesised Metaxin, it could compete for the
import of GR (Study IV). However, the role of Metaxin as an import receptor is still
unclear. In mammals, Metaxin is thought to be part of the SAM complex and is
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involved in the import and assembly of β-barrel proteins. This reduction in β-barrel
import is also seen in Arabidopsis (Study IV) so the deficiencies in GR import could be
a secondary effect. However, the fact that the addition of soluble Metaxin protein was
seen to compete for the import of GR suggests that Metaxin can bind GR. This provides
evidence that dual targeted proteins utilise a different pathway to classical the Tom20
pathwaywhich most mitochondrial proteins utilise.
It is interesting to note that studies on the import components of a number of
plant species suggest that there is no one single plant import apparatus (Study VI).
When the plant mitochondrial import components are compared with yeast and humans,
it is clear that the main translocase subunits (Tom40, Sam50, Tim17:23, and Tim22) are
well conserved throughout all organisms. However, in terms of the receptor subunits
(Tom20, Tom22, and Tom70), there is little or no conservation between yeast and
plants and also very little conservation between different plant species. The plant TOM
complex displays a number of different arrangements in terms of receptors: red algae
only contain a Tom22 like receptor; green algae, moss and lower plants only contain a
Tom20; higher plants such as Arabidopsis and rice contain both Tom20 and OM64
(Study VI). This difference in receptor subunits may be due to the selective pressure of
the cell to differentiate between plastid and mitochondrial targeted proteins. This is
evidenced by the change in plastid targeting signal properties between red algae and
green plants which corresponds to the presence or absence of a full Tom22 receptor. It
has recently been speculated that the dual targeting of a protein is a gain of function
condition derived through gene duplication (Brandao and Silva-Filho., 2010). This
hypothesis is based on the theory that a dual targeted protein started out as a protein
targeted to only one location (mitochondria or chloroplast) and through a gene
duplication event, became subsequently targeted to both organelles and subsequently
lost the single organelle targeted protein (Brandao and Silva-Filho., 2010). If all dual
targeted proteins were once single organelle targeted proteins, it is tempting to speculate
that the evolution of dual targeting goes hand in hand with the diversity of the
mitochondrial import receptors. A better understanding of the evolutionary history of
dual targeted proteins may help to test this theory.
7.3 Reasons for dual targeting
One obvious and outstanding question is why are proteins dual targeted? It is
well known that organelles, such as mitochondria and chloroplasts, share many common
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125
enzymatic steps and the majority of these steps are carried out by location specific
isoforms. So what is the role of dual targeted proteins? Dual targeting does not seem to
be a way of limiting gene numbers in the nuclear genome because organelles also
contain location specific isoforms of dual targeted proteins. A good example is
evidenced with organelle RNA polymerases in Arabidopsis, which contains three
isoforms; one mitochondrial, one chloroplastic, and one dual targeted (Hedtke et al.,
1997, 2000). Rice only contains two; one of which is mitochondrial and the other is
chloroplastic (Kusumi et al., 2004). When the same proteins were analysed in the moss,
Physcomitrella, both were found to be dual targeted to mitochondria and chloroplasts
(Richter et al., 2002). However, conflicting views remain on whether these proteins are
truly dual targeted (Kessler et al., 1994). Therefore, it appears that similar proteins are
not always dual targeted in different species of plants, further confounding the reason as
to why some, but not others are dual targeted. In Arabidopsis inactivation of the dual
targeted RNA polymerase leads to alterations of mitochondrial, but not plastidial,
functions (Kuhn et al., 2009). Thus, dual targeting of proteins may allow specialisation
of function for particular proteins.
One reason for dual targeting that has been proposed, is that it may be a form of
intra-organellar communication. Targeting the same protein to two organelles at the
same time means that these organelles are at least capable of carrying out the same
functions in a co-ordinated manner. This is particularly relevant to plant cells, as they
contain two organelles with their own genome, the replication and/or expression of
which may need to be co-ordinated. Another reason for dual targeting is that new
functions and/or roles can be gained for a specific protein. A good example of this is
Mia40 from Arabidopsis, which was shown to target and accumulate in both
peroxisomes and mitochondria (Study V). This was in contrast to yeast and human
Mia40 proteins, which are solely located within the mitochondrial IMS. The role for
Mia40 in both organelles in plants appears to be similar i.e. in the correct folding of
Ccs1 and subsequently, SOD1. However, by dual targeting Mia40 to peroxisomes as
well as mitochondria, plants have gained an extra function for Mia40 compared to yeast
and humans.
In cases where no organelle specific isoform exists, dual targeting appears to
simply coordinate the same function in both organelles. The majority of known dual
targeted proteins from Arabidopsis are involved in replication, transcription and
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126
translation. This could simply be because the limited number of dual targeted proteins
identified to date are biased towards these roles. Further analysis of the expression
patterns of known dual targeted proteins has revealed that these genes are relatively
static, displaying similar expression levels in most plant tissues and developmental
stages, suggesting that these proteins perform basic, essential functions required in both
mitochondria and plastids (Carrie et al., 2009).
7.4 Future perspectives
Since the discovery of dual targeting in 1995, there have been substantial
developments, with more than 50 proteins described from various plant species that
target to more than one subcellular location. However, our understanding of how, why,
and when this occurs is still very limited. One of the most compelling questions about
dual targeting concerns how proteins are partitioned to each organelle. It has been
observed with some GFP constructs that in different tissues, some proteins appear to
target different organelles at different levels (Study II). The level of GFP fluorescence
for some dual targeted proteins is brighter in plastids compared to mitochondria, while
for other proteins it is equal. Western blots of dual targeted proteins in Arabidopsis
show that more Mia40 is targeted to mitochondria than peroxisomes, and the vice versa
for NDA1 (Studies III and V). A greater understanding of the mechanisms regulating
this partitioning is needed. In yeast, it has been shown that the level of dual targeting of
fumarase is regulated by the metabolites of the glyoxylate shunt cycle (Regev-Rudzki et
al., 2009). It would be interesting to see if different environmental conditions such as
light intensity, temperature or humidity can affect the partitioning of dual targeted
proteins in plants.
While understanding the regulation of dual targeting in plant cells would be a
major breakthrough, it would still leave one big question. That is, why dual target
proteins in the first place? As discussed above, there are a number of theories about why
but there is no definitive answer as yet. A greater knowledge of the evolutionary history
of dual targeting of proteins, combined with the knowledge of the functions of dual
targeted proteins, especially where location specific isoforms exist, will provide the
necessary steps towards understanding why dual targeting of proteins occurs.
References
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