The Function of t h e Qcr7 Protein

of the Mitochondrial Ubiquinol-cytochrome c

Oxidoreductase of Saccharomyces cerevisiae

by

Suzann Malaney

A thesis submitted in conformity with the requirements for the

degree of Doctor of Philosophy

Graduate Department of Biochemistry

University of Toronto

OCopyright by Suzann Malaney 1997

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The Function of the Qcr7 Protein

of the Mitochondrial U biquinol-cytochrome c

Oxidoreductase of Saccharomyces cerevisiae

Doctor o f Philosophy, 1997

Suzann Malaney

Department of Biochemistry, University of Toronto

ABSTRACT

The respiratory chain enzyme ubiquinol-cytochrome c

oxidoreductase (also termed b c l complex or complex III) o f

Saccharomyces cerevisiae contains 1 0 subunits and resides in the

inner mitochondrial membrane (IMM). This multisubunit enzyme

complex is involved in the transfer o f electrons from ubiquinol t o

cytochrome c and in the establishment of an electrochemical

gradient by translocation of protons across the IMM. A previous

study has shown that inactivation of the yeast nuclear gene QCR7,

which encodes subunit 7 (also referred to as Qcr7 protein or 14 kDa

subunit) of the bci complex leads to an inactive enzyme. The bel

complex of the mutant strain lacks holocytochrome b and has

reduced levels of apocytochrome b, the Rieske iron-sulfur protein

(ISP), and the 1 1 kDa subunit. Although this study showed that the

Qcr7 protein is essential for respiration, the exact role of this

subunit is not known.

In the present study 1 have shown by circular dichroism that an

amino-terminal peptide of subunit 7 is a-helical. I have also

studied the effect of mutations in the amino-terminus o f - - th is

protein on mitochondrial targeting, assembly of the bel complex, and

proton translocation. Mu tan t proteins were analyzed by

overexpression of mutated qcr7 genes in the strain YSM-qcr7A,

which contains a disrupted chromosomal QCR7 gene, and by

comparison t o Qcr7 protein from a YSM-qcr7A strain in which the

wild type QCR 7 gene is overexpressed. Respiration proficiency and

the activity of the bci complex were monitored. Based on these

preliminary data, strains expressing mutated proteins that lacked

the N-terminal 7, 10, 14, and 20 residues (after Met-1) of subunit 7

referred to as Qcr7p-~7, -AI 0, -a14, and -a20, respectively, and

strains expressing versions o f Qcr7p-A7 that contained point

mutations RI OK, D l 3V, and R i OI /G lZV were chosen for further

study.

Al1 the mutated versions of the Qcr7 protein, with the

exception of Qcr7p-al0 and Qcr7p-a7(D13V), are present in the

mitochondria (frorn cells grown a t 300C) a t reduced levels of

approximately 55% when compared to Qcr7 protein from the strain

overexpressing wild type QCR7. In contrast, Q c r 7 p - ~ l O is not

detectable and Qcr7p-~7(013V) is present a t wild type levels. This

may implicate the Qcr7 protein amino-terminus in import or in

conferring stability t o the protein. The activity of complex III in

mitochondria from strains with Qcr7p-a7 and Qcr7p-a7(Rl OK) was

normal. In contrast, strains overexpressing qcr7 genes encoding

Q c r 7 p - ~ l O, Qcr7p-A14, Qcr7p-~20, Qcr7p-~7(D13V), and Qcr7p-

A ~ ( R 1 OVG12V) were respiration-deficient. Western blot analyses

from this latter group of mutants indicate that the bcl complex in

respiration-deficient cells displays a significant variation of

i i i

reduced levels of the 11 kDa subunit, as well as combined

intermediate and mature ISP and cytochrome cl . Spectrophotometric

analyses indicate that the amount of holocytochrome b was reduced

in the strain containing Qcr?p-~7(RlOI/G12V) and lacking in the

strain with Qcr7p-d7(D13V). ATP synthesis (an indirect measure of

proton translocation) in mitochondria was comparable t o the wild

type in al1 respiration-proficient strains tested, including the strain

with Qcr7p-A7.

Based on the results of this work, I concluded that the amino-

terminus of the Qcr7 protein is essential for the functional

assembly of ubiquinol-cytochrome c oxidoreductase. In addition to

the proposed function in assernbly, the N-terminal seven residues

may facilitate import into rnitochondria.

DEDICATION

This work is dedicated to my two little daughters Maxine and

Michelle. May it be an inspiration to them throughout their lives.

This work is also dedicated to my husband Robert for his love.

ACKNOWLEDGEMENTS

I would like to thank my supervisor, Dr. Brian H. Robinson.

While in his laboratory for five years I have learned a tremendous

amount about science and life. He has always been supportive,

encouraging, and full of ideas. I thank him for always having an open

door no matter how busy he was. I also thank him for accepting me

into his laboratory and giving me the opportunity to accomplish this

work.

I would like to thank my CO-supervisors Dr. Charles Deber and

Dr. David Williams for al1 their encouragement, support, and

constructive criticism. I would also like t o acknowledge

contribution t o the thesis by Dr. Jacqueline Segall. I am very

grateful to Dr. Bernard L. Trumpower from Dartmouth Medical School

from whom I have gained much knowledge over the years. I would

also like to thank Dr. Peter Lewis, Dr. Reinhart Reithrneier, and Dr.

Shelagh Ferguson-Miller (outside examiner from Michigan State

University) for critically proofreading this manuscript and for many

useful comments.

Somebody who deserves special mention is my husband Robert.

A scientist himself (Astrophysicist), he has been an inspiration and

a hero t o me from the second year of my undergraduate degree.

Without him this work would have never been accomplished. His

dedication as a father and a husband has made it possible for me t o

complete my research.

I would like t o thank rny fellow graduate students and CO-

workers, some of whom deserve special mention. Dr. Frank Merante

and Dr. Sandeep Raha were never too busy t o help out, talk about

science, or play practical jokes. Thank you to Tomoko Myint for her

endless friendship and encouragement. I would also like to thank Dr.

Sandeep Raha, and Dr. Mingfu Ling for critically proofreading this

dissertation and for many useful comrnents. Thank you to Maureen

Waite, our administrative assistant, for easing my work on many

occasions.

I would like to thank my family, especially my mother, for her

never-ending support and for believing in me. Her encouragement

has given me strength. 1 also thank my mother as well as my sister-

in-law Linda for coming from abroad t o babysit.

Thank you Maxine and Michelle for brightening up even those

days on which none of my experiments worked.

TABLE of CONTENTS

DEDIUTON ............................ .. .................................................................................... v

ACKNOW LEDGEM ENTS.. ................... .... . .. .......................................................... .vi

a..

TABLE of CONTENTS.. ............................................................................................ .viit

a.*

UST of FIGURES ....................................................................................................... XIII

......... LIST of TABLES ............. ,... .............................................. .. ............ .. m*

ABBREVWONS ................................................................................................. xvii

CtIAFl€R 1 : Introduction ................................................................................... 1

Components of the mitochondrial respiratory chain .............. ......... 1 O

1. NADH dehydrogenases ....... .. ............................................................................. 1 0

II. Succinate-ubiquinone reductase ............................................................... 1 1

................................................. III. Ubiquinolcytochrorne c oxidoreductase 1 2

vi i i

............................................................... The Rieske ironsulfur protein 1 7

........... What about the supernumerary subunits of complex [Il? 19

................ ................ The supernumerary subunits of complex III .. 20

...................... Subunit 6 may regulate the activity of complex III 20

.................................... Subunit 7: the protein of the current study 21

....................................... Subunit 8: a ubiquinone-binding protein ? 23

Subunit 9 .......................................................................................................... 24

Subunitl O ........................................................................................................ 25

The core proteins .......................................................................................... 25

...................................................... Functional assembly of complex III 26

N . Cytochrome oxidase .......................................................................................... 29

...................................................................................................... V . ATP synthase 31

................................... ...... ............ N ucleo-rnitochondrial interactions ....., .... 33

................. How do mitochondria and the nucleus communicate ? 34

The expression of many complex III subunits is dependent on

........... ....................................... the need for oxidative metabolism .. 36

Do mitochondria exert regulation on nuclear gene

..... products ? ................. ... .... .................................................................. 38 ......................... .................................... Protein import into mitochondria ........ 39

............................................... Import into the mitochondrial matrix 40

The targeting sequence .............................................................................. 43

Cytoçolic chaperones .................................................................................. 44

Receptors in the outer mitochondrial membrane ......................... 46

Pase

The translocation channel and the mitochondrial

....................................................................................................... chaperones 48

.. ............................... lmport into the intermembrane space (IMS) .... 49

................ lmport into the inner mitochondrial membrane (IMM) 5 1

............... lmport into the outer mitochondrial membrane (OMM) 5 2 . . ................................................................................... Objectives of the research 52

................................. CHAPTER 2: Inactivation of the QCR7 gene .... 54

2.1 . INTROOUCTiON .................................................................................................... S S

2.2. MATERIALS AND METHODS ............................................................................. 57

.................................................................................................................... Materials 57

2.2.1 . Disruption of the chromosomal copy of the QCR7 gene ............... 58

2.2.2. Confiming inactivation of the QCR7 gene ......................... .. .......... 60 . . Selection of diploids ............... ... ......................................................................... 60

Northem and Southem bbtting .......................................................................... 60

Isolation of yeast mitochondria ....................................................................... 61

Western analysis ............................... ... ..................................................................... 62

.......... 2.2.3. Raising an antibody in chicken against the Qcr7 protein 63

................... Isolation of anti-Qcr7 antibody from rabbit and chicken -63

2.3. RESULTS .................. .. .................................................................................. 65

Disruption of the QQP7 gene ..................... ..... .................................................. 65 ................. .......*.......*.......... Confirmation of the qcr7 gene inactivation .. 69

2.4. DISCUSSION .................. ... ........................................................................ 73

CHAPTER 3: Mutagenesis of the QCR7 gene ..................................... 74

3.1 . INTRODUCTION ......... .. ........ ....... ...................................................... -75 3.2. MATERIALS AND MmK)DS ............... .... ................................................... 85

Materials ............................................................................................................ ........... 85

...................... .................. .............................. Mutagenesis by PCR ........ ...... 85

Sitedirectecd mutagenesis using the M 1 3 phage ................. .... ............ 88 .

............................................................................................ Enzyme assays ... ....... 89

........................................................................................................... Growth studies 89

............................................................................................... .. ... 3.3. RESULTS ..... ... 91

Characterization of mutants by growth ....................... .. ....................... 91 . . . ................................................................................................. Enzyme activi ties 95

3.4. DISCUSSION ....................................... .. ................................................................ 99

CHAPTER 4: The role of the amino-terminus of the

Qcr7 protein in rnitochondrial targeting. complex III

asse mbly. and proton pumping ........................................................... 102

.................................................................... 4.1 . INTRODUCTION .............. ... 1 03

4.2. MATERUILS AND METHODS ........................................................................ 1 04

Materials ............................................................................................................... 104

Isolation of yeast rnitochondria and Western analyses ........................ 104

.............................................................. ATP synthesis assays ................. ...... 105

............................................................................ Circular Dichrosm spectra 105

Spectra of the cytochrornes .............................................................................. 106 . . . .................................................................................... Co-immunoprecipitation 106

Buffers ............ ... ............................................................................................ A07

Procedures ..................................... ,., ........................................................... 107 . .

Cross-hnking ............................................................................................. 108

4.3. RESULTS ................................... .................. . . . 1 0

Circular Dichroism spectra of amino-terminal peptides ..................... 1 7 0

The amino-terminus of the Qcr7 protein may facilitate import

into mitochondria ................................................................................................... 113

The amino-terminus of the Qcr7 protein is essential for

assernbly of the bel complex ...................................................................... 120 . . .

Co-immunoprecipitations .......... ... .................................................................... 129

Deletion of seven residues from the amino-terminus does not

impair proton pumping ..................................................................................... 131

The strain expressing Qcr7p-~7 is temperature sensitive ................. 132

4.4. DISCUSSION ...........,......................................................................................... 1 3 5

CHAPTER 5: Discussion and Future Directions ........W........... 1 39

DISCUSSION .. ..........,... .............................................................................................. 140

Future Directions ................................................................................................... 158

Confirmation of assembly of the Qcr7 protein N-

terminus with cytochrome b and/or the 11 kDa

subunit and identification of contact sites .................... .. .......... 158

Testing for the involvement of the Qcrï protein

N-terminus in mitochondrial import ............... .... ......................... 161

x i i

LIST o f FIGURES

Figure 1-1

Figure 1-2

Figure 1-3

Figure 1-4

Figure 2-1

Figure 2-2

Figure 2-3

Figure 2-4

High resolution scanning electron micrograph

.................................................................. of a mitochondrion 5

The mitochondrial respiratory chah of S. . . ................................................................................ cerewae. -9

mechanism ............................................................. 1 4

lmport of proteins into the mitochondrial

........................................................ ......................... rnatrix 42

DNA construct for disrupting the QCR7

......................................................................... ...... gene ..... 5 9

Southern blot analysis of the parental strain

W303-1 B and the disrupted strain, qcr7A:LEU2 ...... 68

Northern blot analysis of qcr7d:LEUZ .......... ... ......... 71

Western blot analysis of qcr7A:LEUZ and

qcr7A:LEUZ transformed with the wild type

........................................................................... <Sa77 ge ne.. 72

Figure 3-1 Helical wheelplots and folding pattern of the

13.4 kDa subunit of beef heart mitochondrial

complex III.. ........................................................................ ..77

Figure 3-2 Alignment of yeast Qcr7 protein and its

1 3.4 kDa homolog from beef heart

mitochondrial complex III ................................................. 82

Figure 3-3 Growth on SD agar plate with ethanol/glycerol

and 0.1 % glucose, or SD media plate . -

containing glucose ................................................................ 94

Figure 4-1 Circular Dichroism spectra ..................... .. ......... 1 12

Figure 4-2 Helical wheel projections .................... .. ............... 1 1 4

Figure 4-3 Western blot analyses of mitochondrial

proteins from YSM-qcr7~ strains overexpressing

wild type and N-terminally truncated proteins

Qcr7p-~7, Qcr7p-Al O, Qcr7p-~14.

Qg7pQO .......................................................................... 1 1 6

Figure 4-4 Quantification of complex III subunits in

various Qcr7 mutant strains by densitometry ...... 117

xiv

Figure 4-5 Western blot analyses of mitochondrial proteins

from YSM-qcr7A strains overexpressing

wild type Qcr7, Qcr7p-~7(DI 3V).

Qcr7p-a7(Rl OK), and Qcr7p-~7(RI 01/61 ZV),

respectively .......................................................................... 1 23

Figure 4-6 Difference spectra of the cytoc hromes ................... 1 28

Figure 4-7 Western blot analysis perforrned with

mitochondrial membranes prepared from

YSM-qcr7a (grown a t 37aC) overexpressing

Qcr7p-a7 and the wild type Qcr7

................................................................. ............... protein .. 1 34

LIST of TABLES

Table 1-1

Table 3-7

Table 3-2

Table 4-1

Page

Complex III subunit composition ................................... 13

........................ Prirners used for mutagenesis ..... ........... 87

Sumrnary . Characterization of mutants

according to growth and enzyme activities .............. 98

..................................................................... ATP syn thesis 1 33

xvi

LIST o f A8BREVIATIONS

ATP

BClP

D7-r

EDTA

FlSH

h

IMM

I M S

ISP

kDa

min

mt

NBT

OMM

PAGE

PBS

PMSF

RT

SD media

SDS

adenosine 5'-triphosphate

5-bromo-4-chloro-3-indolyl phosphate-toluidine

ditheiothreitol

ethylenediamine tetraacetate

fluorescence in situ hybridization

hour

inner mitochondrial membrane

intermembrane space

Rieske iron-sulfur protein of complex III

kilodaIton

minute

mitochondrial

p-nitro blue tetrazoliurn chloride

outer mitoc hondrial membrane

polyacrylamide gel electrophoresis

phosphate-buffered saline

phenylrnethylsulfonyl fluoride

room temperature

synthetic def icien t media

sodium dodecyl sulfate

xvi i

CHAPTER 1

Introduction

CHAPTER 1

Introduction

The focus of this study is subunit 7 (also referred to as Qcr7

protein or 1 4 kDa subunit) of ubiquinol-cytochrome c oxidoreductase

(also termed complex III or bc, complex) of the mitochondrial

respiratory chain in Saccharomyces cerevisiae. Complex III is

involved in the establishment of the protonmotive force which is

used to drive ATP synthesis. QCR7, the gene encoding subunit 7, is

nuclear encoded and therefore has to be imported into mitochondria.

Hence, for the functional assembly of complex III, a coordination of

nuclear and mitochondrial genomes is required.

To give a brief account of the topics involved in the study of

the Qcr7 protein, an overview is first given as to where, in the study

of metabolism, mitochondria f i t in. Following this overview is a

general section on mitochondria and a detailed description of the

enzyme complexes in the mitochondrial respiratory chain with an

emphasis on the bc,cornplex and assembly thereof. After that,

nucleo-mitochondrial interactions are discussed and a current

update of mitochondrial protein import is given. The introduction

closes with a brief account of the objectives of this study (a more

detailed account is given a t the beginning of Chapter 3).

If not mentioned otherwise, al1 the information given in this

dissertation and al1 the terminology used are specific t o

Saccharomyces cerevisiae, also referred t o simply as "yeast"

throughout this study.

Ovewie w

Living organisms are not a t equilibrium. Rather, they need a

continuous influx of free energy to maintain order in a universe bent

on maximizing disorder (1). Free energy is derived from the intake of

nutrients, and metabolism is the process that liberates the free

energy so that it can be utilized t o drive the various functions

needed t o sustain life. There are two categories of metabolic

pathways: reactions involved in catabolism and those involved in

anabolism. The driving force for the endergonic reactions involved

in anabolisrn uses ATP and NADPH which are generated by the

exergonic breakdown of a variety of substances during catabolism.

These substances, carbohydrates, lipids, and proteins, are provided

by the nutrient intake of the organism and are catabolized to their

component monomeric units: glucose, gfycerol and fatty acids, and

amino acids, respectiveiy. Subsequently, these metabolites are

converted to the common mitochondrial intermediate acetyl-

coenzyme A which is then further oxidized t o COp by the enzymes of

the citric acid cycle with the concurrent reduction of NAD' and FAD

t o NADH and FADH2. NADH and FADH2 enter the mitochondrial

electron transport chain where they are reoxidized, the final

acceptor of electrons being O*. The complete breakdown of a

substance entering the living system therefore results in the final

products HzO and CO2. The energy generated during this process is

used to drive the synthesis of one of the bioenergetically most

important molecules, ATP. Thus, living organisms are not at

equilibrium, but rather maintain a steady state; despite the intake

of large amounts of water and nutrients, the weight of an adult does

not change significantly over his/her lifetime.

Mitochondria

Mitochondria (Fig. 1-1) constitute the site of eukaryotic

oxidative metabolism. The enzymes of the pyruvate dehydrogenase

complex, the citric acid cycle, as well as those involved in fatty

acid oxidation (in humans), electron transport and oxidative

phosphorylation are al1 located in the mitochondrion. In addition t o

serving as the cell's "power plant", mitochondria, however, also play

roles in the generation of precursors for anabolic pathways and in

calcium homeostasis of the cell (2). Mitochondria, which can occupy

up t o 20% of the cytoplasm of a eukaryotic ceII (31, consist of four

areas, each of which embodies a distinct set of proteins. The

smooth outer mitochondrial membrane (OMM) contains the pore-

forming protein porin which renders the membrane permeable t o

diffusion for a number of molecules. Receptor components involved

in import o f nuclear encoded mitochondrial proteins are also

embedded in this membrane ( 4 - W . The enzymes of the respiratory

chain and oxidative phosphorylation are located in the

intermembrane space (IMS) and the extensively invaginated inner

rnitochondrial membrane (IMM). The number of invaginations

(cristae) in the IMM and sites for oxidative phosphorylation Vary

with the metabolic demand of the cell type. Thus, the surface area

of the inner membrane is larger in a heart muscle cell with a high

Fig. 1-1. High resolution scanning electron rnicrograph of a rnitochondrion (M) typically found in striated rat muscle (x8000).(17*) The cristae are densely packed into the matrix space and show both shelf or plate (short arrow) and tubular (long arrow) morphology. 1, inner mitochondrial membrane (IMM); O, outer mitochondrial membrane (OMM); T, T-tubule.

rnetabolic demand than in a liver cell. The IMM contains more

proteins than the OMM, about 70% by weight, and in beef heart half of

al1 these integral membrane proteins are involved in oxidative

phosphorylation. The remaining 30% of the membrane is lipid in

nature, composed of approximately 1 5 % cardiolipin, 40%

phosphatidylcholine, and 35% phosphatidylethanolamine (1 2 ) . Unlike

the OMM, the IMM is freely permeable only to Op, COZ, and H20 (2).

This limited permeability of the IMM permits the generation of ionic

gradients, but it also calls for a number of transport mechanisms.

The fourth and innermost region of the mitochondrion is the matrix,

a gel-like compartment, that consists of approximately 50% water.

The matrix contains the pyruvate dehydrogenase complex, the

enzymes of the citric acid cycle and fatty acid oxidation, as well as

substrates for the electron transport chain, nucleotide cofactors,

inorganic ions, and the mitochondrial genetic and protein synthetic

machineries (1 3).

Theories as to the origin of mitochondria now favor the

endosymbiosis hypothesis, such that mitochondria contain their own

genetic code, DNA replication and protein synthetic machineries. As

can be seen from fixed sections under the electron microscope,

mitochondrial (mt) DNA may exist in three alternative forrns: closed

circular, linear, or aggregates of large and small circles. The size

of the DNA molecule can Vary extensively from 16 kb in animals to

100 kb in plants (2). Saccharomyces cerevisiae has one of the

largest mt DNA molecules seen in fungi, a circular molecule of 70-

75 kb which is 21 -25 pm long. Yeast m t DNA also contains introns

and intergenic spacers. On the other hand, the circular, 16.5 kb mt

DNA molecule of humans is highly efficiently organized and is

therefore one of the smallest ones, being only 4.8-5.1 pm in length.

Yeast mt DNA encodes 24 tRNAs, as weli as large and small rRNAs;

although yeast mt DNA may encode more than one small and one large

rRNA, only one of each is functional. In addition, yeast rnt DNA also

encodes cytochrome oxidase subunits 1, 11, III, cytochrome b, ATPase

subunits 6, 8 , and 9, RNA maturases, an intron transposition

endonuclease, an RNA component RNase-P-like enzyme and a

ribosome-associated protein (3. '3). The introns of the cytochrome b

gene are believed to encode maturases required for the splicing of

its RNA transcript (14). Some strains of S. cerevisiae have a split

21 S rRNA gene; the intron of this gene encodes a protein, however,

with a function other than RNA splicing ( 2 ) . All genes are

transcribed from the same strand, except for the tRNA for threonine.

Initiation of transcription is marked by the sequence S-atataagta-

3', whereby the last adenosine constitutes the first nucleotide of

the translational initiation codon atg. In yeast, there are 20

different primary transcripts which al1 contain a t least two coding

sequences (1 5).

The simple and highly organized mitochondrial genome of

humans encodes only two rRNAs, 22 tRNAs, and a small number of

proteins o f the respiratory chain and oxidative phosphorylation. The

human mitochondrial genome does not contain any introns or

intergenic spacers, nor does it encode any regulatory elernents. In

al1 animal mitochondria, both strands are transcribed from a single

promoter and the prirnary transcripts contain al1 the information

encoded in each of the two strands. There are no 5' leaders in the

transcripts; however the tRNA genes which are present between

most genes signify the s ta r t and end of a gene (15). From the small

mitochondrial genome in animals it is evident that only a few of the

hundreds of mitochondrial proteins are encoded by mitochondrial

DNA. Hence, most of the respiratory chain enzymes as well as most

of the components of the mitochondrial genetic system such as

mitoc hondrial DNA polyrnerase, mitochondrial RNA polymerase,

ribosomal proteins, and translation factors are encoded by nuclear

DNA (3).

The mitochondrial respiratory chain consists of five enzyme

complexes which are ernbedded in the inner mitochondrial membrane

(Fig. 1-2). Electron transport through complexes III (also referred to

as ubiquinol-cytochrome c reductase or bc, complex) and IV

(cytochrorne oxidase) in S. cerevisiae and complexes I (NADH-

ubiquinone reductase), III, and IV in humans is coupled to vectorial

proton translocation into the IMS, resulting in the establishment of

a proton gradient and subsequent membrane potential. The energy

from this gradient is then used as the driving force for ion

translocation and protein import into mitochondria, as well as for

ATP synthesis, which is catalyzed by complex V, the F,F, ATPase (2.

12).

There are some differences between the mitochondria of yeast

and those of humans and many of these differences are the result of

H I H+ "extemal" NADH H+ H+ H+

dety dmgenzse H+ H+ 1 MS NADH kf +

"internai" NADH drhydrognase f

FADH2

Fig. 1-2. The mitochondrial respiratory chain of S . cerevisiae. Sketch of the four complexes of the mitochondrial respiratory chain in yeast: succinate dehydrogenase (complex II), u biquinol-cytochrome c oxidoreductase (complex III or bc, complex), and cytochrome oxidase (complex IV), as well as the ATPase (complex V). Arrows indicate the order of electron flow and concurrent proton ejection into the IMS by complex III and the final electron acceptor, complex IV. Protons are transported back across the IMM against the electrochemical gradient by the ATPase, complex V.

regulation of the mitochondrial and cytosolic redox balance as

expressed by the NAD/NADH and NADP/NADPH redox ratios. For

example, unique to yeast mitochondria are the L-lactate:cytochrome

c oxidoreductase, cytochrome c peroxidase, and the external and

internal NADH dehydrogenases. These NADH dehydrogenases are

involved in the oxidation of cytosolic NADH and thus there is no need

for shuttle systems which transfer NADH from the cytosol t o the

mitochondria. Furthermore, aspartate arninotransferase is only

present in the cytosol, hence yeast do not have a functional malate-

aspartate shuttle; this, also, is probably a result of their ability t o

oxidize externally added NADH. A fatty acidhalate-citrate shuttle

is not necessary, because of the absence of p-oxidation of fat ty

acids in most yeast mitochondria (2. '6. ' 7 ) .

Components of the mitochondrial respiratory chain

1. NA DH dehydrogenases

In humans, respiratory chain complex I is the largest of al1 the

enzyme complexes and consists of at least 42 subunits (18) with

many of the subunits having been discovered in recent years. In S.

cerevisiae on the other hand, this enzyme complex does not exist.

However, yeast have two NADH dehydrogenases which are located in

the IMM. One of these is an "external" type of mitochondrial NADH

dehydrogenase, which can oxidize externally added NADH. This is a

function that mammalian cells do not possess. The other is an

"internal" NADH dehydrogenase. This enzyme consists of one subunit

(mw=53000) which contains FAD and reacts with ubiquinone. This

molecule carries out the oxidation of NADH which is generated in the

matrix by the citric acid cycle. Both types of NADH dehydrogenases,

unlike the human NADH-ubiquinone reductase, are insensitive t o the

inhibiton rotenone and piericidin and neither one of them is coupled

t o proton translocation across the IMM (16, 17). Under conditions of

stawation for carbon and nitrogen, however, S. cerevisiae is capable

o f synthesizing a NADH dehydrogenase that is involved in the

establishment of a proton gradient. This dehydrogenase oxidizes

intramitochondrial NADH and is insensitive to rotenone (2. 16).

The yeast S. cerevisiae also contains a flavoprotein-containing

NADH dehydrogenase that is embedded in the OMM. This enzyme,

which is antimycin insensitive, catalyzes the oxidation of

cytoplasmic NADH and is overexpressed in a ubiquinone-deficient

mutant (1% 20). This NADH dehydrogenase is a component of an outer

membrane electron transfer chain in which electrons flow via a

cytochrome b5.to cytochrome c.

II. Succina te-ubiquinone reduc tase (complex II)

Succinate-ubiquinone reductase, also known as complex II, is

located on the matrix side of the IMM and catalyzes the oxidation of

succinate and the reduction of ubiquinone to ubiquinol (Fig. 1-2). In

humans, complex II is composed of four subunits: a 70 kDa

flavoprotein that contains covalently bound FAD, a 27 kDa iron-

sulfur protein (ISP) with three iron-sulfur clusters, as well as a

15.5 and a 13.5 kDa subunit which are membrane-bound and contain

b-type hemes. This enzyme complex, which consists solely of

nuclear encoded subunits, does not transfer protons into the IMS and

is thus not involved in the establishment of the proton gradient.

This is probably why this complex has not been studied as

extensively as complexes III and IV in yeast and complexes 1, III, and

IV in humans (21).

Ill. Ubiquinol-cytochrome c oxidoreductase (complex 111, bc, complex)

Complex III, a homodimer (1621, accepts electrons from

complexes I and II via the electron shuttle ubiquinol and catalyzes

the reduction of cytochrome c (Fig. 1-2). In human and beef heart

mitochondria, complex III consists of 11 subunits (228 23) and a

recent documentation on the preliminary crystallization of the beef

heart mitochondrial complex predicts 1 3 transrnembrane helices for

each monomer (162). SO far, only 10 subunits have been discovered

for yeast complex III (Table 1-1 ) (2. 24). Hence, these complexes

contain six to eight additional polypeptides than the homologous

enzyme complexes of some other organisms. The chloroplast and

cyanobacterial bsf complexes have four subunits and the complex

from P. denitrificans has a mere three polypeptides (25-27). Although

the number of subunits varies widely in al1 these comparable enzyme

complexes, they al1 contain the same number and kinds of prosthetic

groups. There is a b-type cytochrome which contains two hemes, a

cytochrome c l or f which contains one heme, and the Rieske iron-

sulfur protein (ISP) which contains a [ZFe-2S] ferredoxin. The

subunits containing prosthetic groups are also referred to as the

catalytic core. The mode of electron transfer is believed to be

according to the Q-cycle mechanism (28-30) (Fig. 1-3). In addition to

the subunits containing redox centers (cytochrome b, cytochrome c,,

I Table 1 -1 . COMPLEX III SUBUNIT COMPOSITION

I - -

I YEAST BEEF HEART Subunit Daltons

Core 1 Core 2

Subunit Daltons

Core Proteins 48,225 38,714

Core 1 Core 2

Catalytic Su bunits C Y ~ b 43,633 CYt Cr 27,419 ISP 23,349

Supernumenry Subunits

Cyt b CYt G ISP

17 kDa (QCR6) 14,513 1 4 kDa (QCRT) 14,561 1 1 kDa (QCRB) 1 1,000

7.2 kDa (QCR9) 7,262 8.5 kDa (QCR70) 8,492

Hinge 9,175 13.4 kDa 13,389 9.5 kDa 9,507 DCCD 7,998 7.2 kDa 7,189 6.4 kDa 6,363

Center

Center

Fig. 1-3. Q-cycle rnechanism as adapted from Trumpower (28).

The scheme shows the pathway of electron transport through the bc, complex with the reaction order through the four redox centers indicated by numbers (1) through (4). In step (1) ubiquinol is oxidized a t center P whereby one electron is transferred from ubiquinol t o the iron-sulfur protein (ISP). The newly generated ubisemiquinone anion a t center P immediately reduces the cytochrome b-566 heme in what is referred to as oxidant-induced reduction. This first step results in the deposition of two protons on the positive side of the membrane, center P. In step (2) the electron from the ISP is transferred to cytochrome c l which subsequently transfers it to cytochrome c of complex IV. In step (3) the electron from the b-566 heme is transferred to the higher potential b-560 heme against the membrane potential. In step (4a) ubiquinone is reduced to the relatively stable ubisemiquinone anion a t center N. Step (4b) occun in the second round o f the cycle and in this round the b-560 heme reduces the ubisemiquinone anion to ubiquinol with the consumption of two protons from the matrix side of the membrane. Thus one complete Q-cycle deposits four protons ont0 the positive side of the membrane, and transfers two electrons to complex IV, thereby reducing two molecules of cytochrome c.

and ISP), there are two core proteins and five so-called

supernumerary subunits (encoded by the genes QCR6, QCR7, QCRB,

QCRS and QCR 70 in yeast).

vtochrome b

Cytochrome b, the only mitochondrially encoded subunit of

complex 111 ( 3 4 represents the Iiydrophobic core of the complex with

eight predicted membrane-spanning a-helices. Its primary structure

is well conserved among various organisms. The two hemes, b566

and b562? are located on opposite sides of the membrane, one at the

positive side, center P (also referred to as center "O" for proton

output), and one towards the negative matrix side, center N (also

called center "i" for proton input). This is in agreement with the Q-

cycle mechanism and the eight membrane-spanning helix structure

is therefore the accepted model, as opposed to the nine membrane-

spanning helix structure proposed in earlier studies (2). The eight

membrane helix model is also consistent with a number of studies

involving mutants that are resistant to inhibitors (31). Two pairs of

conserved histidine residues are the ligands to the two heme groups;

each heme is bound to one histidine in the second helix and one in the

fourth helix. Mutants that lack cytochrome b are devoid of complex

III enzyme activities and display low steady-state levels of the ISP

as well as of the 14 kDa and 1 1 kDa supernumerary subunits. The

rate of synthesis of these three subunits is not affected in

cytochrome b mutants. Rather the low steady-state levels are

believed to be the result of a faster turnover rate since cytochrome

b, the 14 kDa and the 11 kDa subunits have been shown to form a

subcomplex for which the ISP has a high affinity (2.31-34).

Cytochrome b has been shown to be involved in ubiquinone-

binding by photoaffinity labeling of inhibitors. lnhibitors of complex

III such as hydroxy quinoline N-oxide (HQNO), diuron, 6-undecyl-5-

hydroxy-2,3-dioxobenzothiazole (UHDBT), stigmateIIin, and

myxothiazol which are ubiquinone, ubiquinol, or ubisemiquinone

analogs bind cytochrome b a t center P or N. Cytochrome b by itself

is believed t o form the binding site for ubiquinone at center N;

therefore inhibitors of this site, such as antimycin, block the

reoxidation of cytochrome b and destabilize the bound

ubisemiquinone. On the other hand, the ubiquinone binding site a t

center P is formed jointly by cytochrome b and the ISP; hence,

inhibitors of this site, such as myxothiazol, block reduction of the

ISP and also prevent reduction of cytochrome b in the presence of

antimycin (281 32) (see Fig. 1-3).

çytochrome c,

Cytochrome c i , a protein with a molecular weight of 27,419 is

another heme-containing subunit of the bc, complex. All cytochrome

cl proteins studied so far contain the consensus sequence CXXCH a t

residues 40-44 of the mature protein. This is a characteristic of

protoporphyrin IV proteins in which the heme is liganded to a

histidine residue (2. 35). In addition, a conserved methionine a t

position 164 has been proposed as the sixth ligand t o the heme iron.

Although cytochrome c l is overall hydrophilic, it has a nonpolar

stretch of 15-20 amino acids near the carboxy terminus. It has been

suggested that this region forms an a-helix which anchors the

protein in the membrane (35). The remaining part of the protein,

including the catalytic center, is believed t o be located in the IMS

(162). Cytochrome cl is a nuclear encoded subunit that is synthesized

with a bipartite signal sequence (two amino-terminal leader

sequences arranged in tandem), 61 amino acids long. Two different

mechanisms have been suggested to explain the targeting of this

protein into its final destination in the IMS (36 .37) (see below).

The Rieske iron-sulfur protein

The Rieske ISP with a calculated molecular mass of 23,349

daltons and a length of 215 amino acids is the third subunit of

complex III containing a redox center. Just like cytochrome c l , the

ISP is nuclear encoded and synthesized with a bipartite signal

sequence 3 0 residues long (MLGIRSSVKTCFKPMSLTSKRLISQSLLAS)

(381 39). Its catalytic center is located in the aqueous environment of

the IMS (162) and is linked to the inner membrane by a hydrophobic

anchor. Proteolytic cleavage a t a site just downstream from the

putative membrane spanning helix of the ISP in E. coli and R.

sphaeroides renders the protein water-soluble (40).

The ISP contains a [2Fe-ZS] ferredoxin with a very high

midpoint potential which is liganded to the two histidines and two

of the cysteine residues in the following conserved sequences: CPCH

and CTHLGC. Due to its high redox potential the ISP is the primary

electron acceptor during hydroquinone oxidation and is thus involved

in what is often referted t o as "oxidant-induced reduction" of the

lower potential heme of cytochrorne b (refer t o the Q-cycle

mechanism, Fig. 1-3). Like cytochrome b, the ISP binds stigmatellin

and ubiquinone analogs. Hence, it has been suggested that both

cytochrome b and the ISP interact to form the binding site for these

inhibitors (2, 28,38, 39,41,42).

Results from mutational analyses of the ISP suggest that

substituting the conse~ed residues that constitute the ligands to

the [ZFe-2S] cluster does not prevent processing of the precursor to

the mature polypeptide, although a slight increase of incompletely

processed iron-sulfur apoprotein can be detected in the mutants.

Mutagenizing the ligands, however, prevents the [ZFe-2S] cluster

from being inserted into the apoprotein and, although it does not

prevent assembly of the bc, complex, the iron-sulfur apoprotein is

more easily lost upon purification of the complex. Earlier studies

indicated that when the gene encoding the ISP is deleted the

cytochrome b hemes are distorted (39) . However, a mutational

anaiysis performed by Graham and Trurnpower (42) seems to indicate

that the simple presence of the iron-sulfur apoprotein is sufficient

to stabilize the environment of the b-hemes resulting in near wild

type levels of this holoenzyme. Based on these results it was

suggested that the insertion of the [2FeW2S] cluster is the final step

in the biosynthesis of complex III. Furthermore, it was suggested

that the iron-sulfur apoprotein itself is involved in stabilizing the

heme environment of cytochrome b and that the [ZFe-2S] cluster is,

in turn, invoived in stabilizing the attachment of the ISP t o the

complex (42).

What about the supernurnerarv subunits of com~lex III?

When comparing eukaryotic and prokaryotic bclcomplexes, it

becomes evident that some bacterial isoenzymes that only contain

the three catalytic subunits (cytochrome b, c,, and the ISP) perform

the same function as their eukaryotic counterparts that contain as

many as 11 subunits. This raises the question as to the functional

importance of the so-called supernumerary subunits. Two models

are used t o rationalize their presence. In the first model, a11

primordial organisms are believed t o have contained supernumerary

subunits. Subsequently, as bacteria and mitochondria branched off

from one another during the course of evolution, bacteria, being

more efficient, lost these subunits whereas mitochondria retained

them. From this model it follows that the supernurnerary subunits

are redundant and probably do not serve any function. The second

model States that none of the primordial organisms contained

supernumerary subunits, but rather that they were acquired during

the course of evolution by mitochondria as a result of morphological

differences between bacteria and mitochondria. This latter model

implies that the supernumerary subunits serve a function since why,

otherwise, would they have been acquired during the course of

evolution? All the yeast genes encoding the supernumerary subunits

of complex III have been inactivated and found to be necessary for

the integrity and functioning of the compiex (see below). Hence, the

knowledge that is available today supports the second model

according to which the supernumerary subunits are believed to serve

a function.

The SuDernurnerary subunits of com~lex III

abun i t 6 may reglate the activity of complex III

QCR6 is the yeast gene that encodes the well-conserved, highly

acidic polypeptide of 14.5 kDa that is actually referred to as the 17

kDa subunit due to its migration on SDS-PAGE (it is also known as

subunit 6). This nuclear encoded subunit is the only polypeptide of

the supernumerary subunits that is synthesized with a cleavable, N-

terminal targeting sequence, 25 amino acids long (2. 43). The Qcr6

protein contains an acidic stretch of 24 amino acids, is largely a-

helical. and has a domain which resembles the Ca2' binding domain of

yeast calmodulin (44). However, an involvement of this protein in

Ca2' binding has not been demonstrated to date. The only hydrophobic

region of the protein is at the carboxy-terminus and this region is

postulated to anchor the subunit to the inner membrane. Subunit 6 is

homologous to the "hinge protein" of the bcl complex in beef heart

which has been implicated in promoting ionic interactions between

cytochrome c and cytochrome c l , thereby facilitating electron

transfer f rom the bc, cornplex to cytochrome oxidase (45).

Deleting the QCR6 gene gives rise to a strain that has 50%

residual ubiquinol-cytochrome c reductase activity and maintains

the ability t o grow on non-fermentable carbon sources (46). Schmitt

e t al. have suggested that this deletion causes a conformational

change of the complex that results in "silencing" half of the

reduction sites of the bel dimer. This led t o the belief that the 17

kDa protein is involved in regulating the activity of the complex in

vivo during conditions when the requirement for ATP is low. Hence,

under such conditions a signal must therefore be present in vivo that

rnirnics the action of the deletion. In summary, these gene deletion

studies have shown that subunit 6 is not necessary for assembly of

complex 111 (46). On the other hand, studies performed in which the

QCR6 gene was disrupted, rather than deleted, resulted in a complex

that lacked any detectable enzyme activity and holocytochrome b

(47). This was believed to be due to the existence of an aberrant copy

of the 17 kDa protein that was being synthesized. This non-

f unctional polypeptide supposedly prevented the complex from

assembling by binding either to cytochrome cl or the Qcr9 protein

thereby removing these subunits from the assembly pathway and

preventing further assembly. Overexpressing the Qcr9 protein could

partially correct for this defect by binding the aberrant Qcr6 protein

and preventing it from removing cytochrome c l from the assembly

pathway. From these results it was concluded that subunits 6 and 9

interact with cytochrome cl to form a subcomplex (47).

Subunit 7: the protein of the current studv

QCR7 is the nuclear gene encoding the so-called 14 kDa subunit

(also referred to as subunit 7 or Qcr7 protein) which has a molecular

weight of 14,561 and is the focus of the current study. Subunit 7 is

a largely hydrophilic protein with a hydrophobic stretch from

residues 19-38. The QCR7 gene has been cloned and sequenced; the

deduced protein was found to be homologous to the 13.4 kDa subunit

6 o f the beef heart mitochondrial bc, complex (23s 339 48). The

upstream sequence of this gene has elements similar to a CAAT and

TATA box, which are important for transcription initiation in

humans. However, this promoter region lacks motifs for high level

expression in yeast leading to a low abundance RNA transcript (48).

Previous inactivation of the gene encoding subunit 7 indicated

that the 14 kDa subunit is crucial for complex III function (33). The

mutant strain is respiration-deficient and does not grow on non-

fermentable carbon sources. The mutant displays low steady-state

levels o f the 11 kDa and ISP subunits. In addition,

spectrophotornetric analyses indicate that holocytochrome b cannot

be detected and Western blot analyses show that apocytochrome b

levels are far below those in the wild type. As RNA transcripts of

the 11 kDa subunit and the ISP are present at wild type levels, the

results indicate that the low steady-state levels of the above

proteins are likely to be due to a post-transcriptional effect. Hence,

the pet- phenotype (the phenotype resulting from a respiratory chain

defect) seen in the mutant in which the QCR7 gene has been

inactivated, is most iikely due to an inability of the bci complex t o

assemble, which in turn results in degradation of the subunits

associated with the Qcr7 protein. Based on these results Schoppink

e t al. suggested that the 14 kDa subunit associates with cytochrome

b, the 11 kDa subunit, and possibly the ISP t o form a subcomplex

before further assembling into a functional enzyme complex. A

study performed by Hemrika e t al. (494 in which the C-terminal 12

residues were replaced by the 3 residue segment L-A-D, resulted in

complex III activity that was reduced by approximately 60% when

compared to the wild type, whereas the turnover number was normal.

This mutant also displayed lowered levels of holocytochrome b, the

11 kDa subunit, and the ISP. In the same study, a mutant was

constructed in which the C-terminal 31 residues were replaced by

the sequence D-L-Q-P-S-L-L-1-D. This mutant displayed a phenotype

similar t o the strain in which the QCR7 gene is inactivated. The

authors concluded from this study, that the C-terminus of the 14

kDa subunit is involved in the formation of a functional enzyme

complex. However, as the authors did not have an antibody that

recognized the truncated 14 kDa subunits, it cannot be ruled out

from this study that the decreased levels of assembly are in fact a

secondary effect resulting from decreased import of the 14 kDa

su bunit.

Subunit 8: a ubiauinone-bindina rotei in? QCR8 is the nuclear gene encoding a polypeptide of 11 .O kDa

which is usually referred to as the 11 kDa subunit (also referred t o

as subunit 8) of the bc1 complex (50, si). The amino acid sequences of

this subunit and its 9.5 kDa beef heart mitochondrial counterpart,

which has been implicated in ubiquinone-binding (521, are not

significantly similar, however their predicted secondary structures

are very similar (53). Mutagenesis of an aromatic region of the 11

kDa subunit has indicated that this region may be involved in the

assembly of a functional enzyme and in ubiquinone-binding a t center

P (50, 54, 55). Deletion of the QCR8 gene leads to low steady-state

levels of the 14 kDa subunit as well as the ISP and cytochrome b, the

latter being detected by absorption spectra. These results are in

agreement with the gene deletion studies of subunit 7 and further

strengthen the hypothesis of a subcomplex consisting of the 11 kDa

and 14 kDa subunits, and cytochrome b. Since deletion of the gene

encoding the ISP does not affect assembly of the bel complex, it is

not understood a t which stage the ISP associates with the above-

mentioned subunits, and whether it is indeed an inherent part of this

su bcomplex.

Subunit 9

QCR9, the gene encoding subunit 9 has a molecular weight of

7,300 and displays 56% identity to its beef heart counterpart.

Deletion of this gene results in a strain that is respiration deficient

with a residyal activity of less than 5% although complex III is still

assembled ( 5 6 ) . Upon further examination, the level of cytochrome cl

was found to be normal; holocytochrome b levels were found to be

decreased in the mutant strain when compared to the wild type.

Mature ISP was present although without the [ZFe-2S] cluster and,

as a consequence, the pre-steady-state rate of reduction of

cytochrome c l was decreased. Based on these results, it was

suggested that subunit 9 interacts with the ISP and the cytochrome

b domain involved in binding the lower potential b-566 heme at

center P. The absence of subunit 9 is therefore believed to invoke a

conformational change of the ISP which prevents the insertion of the

[ZFe-ZS] cluster. This conformational change of the ISP would then

cause a distortion of the b-566 heme environment and thereby alter

the interaction of the ISP with this low potential cytochrorne b

heme. This is so far the only described case in which the absence of

a supernumerary subunit results in respiration deficiency with the

bel complex still being assembled (44.56).

ubunit 10

The most recent subunit that has been discovered is the 8.5

kDa subunit 10 (8,492 daltons) that is encoded by the nuclear gene

QCRIO (24). This polypeptide is the homologue of the 6.4 kDa subunit

11 in the beef heart bcl complex. The two proteins display similar

secondary structures and their amino acid sequences are 51%

similar. Deletion of the QCRlO gene alone does not affect growth on

non-fermentable carbon sources and the mutant strain has a residual

complex III enzyme activity o f 40%. However, deleting both QCR 7 O

and QCR6 gives rise to a strain with a decreased growth rate on non-

fermentable carbon sources. Furthermore, when the bcl complex of

the QCRl O deletion mutant is purified, the ISP is lost. From these

results it was suggested that subunits 6 and 10 rnay be genetically

linked, and that the Q C R 7 O protein may contribute to a tighter

association of the ISP with cytochrorne b (24).

The core proteins

Core proteins 1 and 2 are the largest subunits of ubiquinol-

cytochrome c oxidoreductase and account for 50% of al1 the protein

in the complex. They are both encoded by nuclear genes and have

cleavable N-terminal signal sequences 17 and 16 amino acids long,

respectively. In contrast t o al1 the other subunits of the complex

the core proteins are nearly always present at wild type levels in

strains in which the other subunits of complex III are absent as a

result of gene inactivations. Deletion of the gene encoding core

protein 1 results in a pet- phenotype. The mutant strain does not

grow on non-fermentable carbon sources. Core protein 1 of

Neurospora crassa bc1 complex has been found to be the previously

identified processing enhancing protein (PEP) which stimulates the

activity of the matrix processing peptidase (MPP) in N. crassa (57).

In S. cerevisiae, core protein 1 is homologous, but not identical to

the PEP of yeast (58. 59). Deletion of the gene encoding core protein 2

results in a strain that grows slowly on non-fermentable carbon

sources. Mutants lacking both core proteins have low residual

enzyme activity, but the turnover number is the same as in the wild

type. Hence, the core proteins are not involved in the catalytic

activity of the bel complex, but rather increase the efficiency of the

assembly process (2).

Functional assemblv of complex III

All the research that has been performed on the bc, complex t o

date suggests that three subcomplexes are formed prior to assembly

of a functional enzyme complex. One of the subcomplexes is

believed to consist of cytochrome b, the 14 kDa and 11 kDa subunits.

The reason for the proposed association of these three subunits is

their post-translational interdependence, which has been

demonstrated by the study of strains in which the respective

subunits were missing or mutated (33. 49. 55, 60-63). Although steady-

state levels of the ISP are also reduced in mutants lacking either

cytochrorne b, the 11 kDa or the 14 kDa subunit, the ISP is not

believed to be a component of the cytochrome b subcomplex. This

conclusion is based on findings that a mutant strain containing a

deletion of the gene encoding the ISP does not contain lowered

steady-state levels of any complex III subunits other than of the

ISP; in addition, the insertion of the ISP is believed t o be the last

step in the biosynthesis of complex 111 (56? 60, 64). Hence, the lowered

steady-state levels of the ISP are more likely t o be a secondary

effect resulting from degradation of this protein when its

neighboring subunits are not present. Two nuclear gene products,

encoded by CBP3 and CBP4 in yeast, have been proposed to be

involved in assembly of the bc, complex or maturation of cytochrome

b (directly or indirectly) (61. 62). More specifically, the Cbp3 and

Cbp4 proteins are suggested to stabilize or assemble the cytochrome

b subcomplex since cbp3 and cbp4 mutants display reduced levels of

cytochrome 6, the ISP, and the 11 kDa and 14 kDa subunits. This

might occur by stabilizing cytochrome b, so that it remains in a

competent state for the insertion of hemes or the association with

the 14 kDa and 11 kDa subunits. This notion is consistent with the

belief that maturation of cytochrome b occurs after the formation of

the cytochrome b subcomplex or even after the assembly of this

subcomplex into a stable quarternary complex of which the

composition is unclear a t this point (60).

A second subcomplex that is formed is believed to consist of

core proteins 1 and 2 (47. 57, 60. 65). Contrary to the cytochrome b

subcomplex, this subcomplex is quite stable ( 2 ) . Hemrika e t al. (49)

have proposed that the charged C-terminus of the 14 kDa subunit

associates with this subcomplex consisting of core proteins 1 and 2

and that this association renders the cytochrome b subcomplex

protease resistant. This model is in agreement with previous

results which show that steady-state levels of the 14 kDa subunit

and cytochrome b are decreased in the absence of core protein 2 due

t o an increased sensitivity t o proteases ( 6 6 ) . The model is

furthermore consistent with the more recent prediction of the

topology of the 14 kDa subunit, that not only the N-terminus of this

subunit faces the matrix (661, but also the C-terminus ( 6 7 ) . This is

important because the core proteins are located on the matrix side

of the IMM with a large fraction protruding into the matrix (57).

The third subcomplex is believed to consist of cytochrome c i ,

the 17 kDa and the 7.2 kDa proteins. The prediction of the formation

of a subcomplex composed of these three subunits is based on

findings that the homologues of the yeast 17 kDa and 7.2 kDa

subunits have been cross-linked t o cytochrome c, in beef heart (6s).

Further evidence for the formation of this subcomplex is given by

studies which indicate that the association of the 17 kDa subunit

with cytochrome c, renders the latter resistant t o proteases and

stimulates the interaction between cytochromes c , and c ( 45 ) .

According to Schmitt and Trumpower (47) the 17 kDa and 7.2 kDa

subunits associate with each other first, before combining with

cytochrome c, into a subcomplex of intermediate stability. This

subcomplex then associates with the partially assembled complex

III, consisting of the cytochrome b and core protein subcomplexes, to

form a stable oligomeric complex without the ISP. Insertion of the

ISP completes the assembiy pathway of ubiquinol-cytochrome c

oxidoreductase ( 4 7 , W

Much of the information that led to the prediction of the three

subcomplexes discussed above was based on the varying steady-

state levels o f the supernumerary subunits of complex III in

different mutant strains. In most cases, it was concluded that

lowered steady-state levels were due to an improperly assembled

subcomplex or bc,complex. Hence, the subunits that are lowered are

believed to assemble with the mutated or absent protein. This

prediction is probably true in most cases. However, it cannot be

ruled out that in some cases, in which a protein is mutated rather

than absent, the mutated protein is more susceptible to proteolysis

and that in these instances the lowered steady-state levels of other

subunits are the result of a secondary effect (68-71). As none of the

proteases (except the one responsible for the turnover of the F,P

subunit of the ATPase) involved in the turnover of the nuclear

encoded subunits o f the respiratory-chain enzyme complexes are

known a t the present time, this subject remains open to speculation

(68).

IV. Cflochrome oxidase

Cytochrome oxidase (also referred to as complex IV and COX),

is the terminal enzyme complex of the mitochondrial electron

transport chain (Fig. 1-2). It catalyzes the oxidation of cytochrome

c and the irreversible reduction of Oz t o HrO. In the crystal

structure of cytochrome oxidase from Paracoccus denitrificans ( l s g ) ,

which contains 22 prirnarily helical membrane-spanning segments,

lwata e t al. have identified two possible proton transfer pathways.

These are for the scalar movement of protons which is involved in

the reduction of Oz t o H20, and the vectorial movement of protons

which is involved in the establishment of the protonmotive force in

the IMS ( '59). In the crystal structure of the 13 subunit beef heart

cytochrome oxidase by Tsukihara e t ai. (1 681, three networks capable

of relaying protons were identified; two for the vectorial movement

of protons and one for the scalar movement of protons. Of the 13

subunits of cytochrome oxidase (721, only the three largest ones are

encoded by mt DNA and these constitute the catalytic core. These

are cox 1, 2 , and 3 and they are strikingly similar in beef heart and

in the three subunit-containing enzyme complex of P. denitrificans

as well as to their counterparts in mitochondria of other organisms.

Although cox 1, 2, and 3 are referred to as the catalytic core, only

cox 1 and 2 contain redox centers. Cox 1, contains Cu.and hemes a

and a3, which in beef heart mitochondria are in perpendicular

orientation t o the plane of the membrane. Herne a3 and Cu.

constitute the binuclear center a t which O2 is reduced to H20. Cox 2,

which contains Cu., and cox 3, of which the function is unknown,

associate with the transmembrane region of cox 1 without any

direct contact with each other (168). In the beef heart mitochondrial

cytochrome oxidase cox 1 is mainly embedded in the membrane with

12 transmernbrane helices, whereby the N- and C-termini o f cox 2

are both located on the cytosolic side. All of the 10 nuclear encoded

subunits of beef heart cytochrome oxidase were found to contain

transmembrane a-helices, most of which are neither vertical t o the

plane of the membrane nor parallel to each other (168). In yeast,

nuclear encoded subunits 7 and 7a have been implicated in assembly

of complex IV (731, while subunits 4 and 8 seem to be involved in the

stability of the enzyme (74, 75) . The order of electron transfer

through cytochrome oxidase is from cytochrome c, the primary redox

center, to the Cu, center, then to heme a, and then t o the binuclear

center.

IL A TP synthase

The ATP synthase or complex V (Fig. 1-2) is the enzyme

complex that catalyzes the phosphorylation of ADP t o ATP (769 77).

The complex is composed of three parts: a water-soluble FI portion

("headpiece"), a detergent soluble Fo portion ("basepiece"), and a

"stalk". The role of the Fo portion is to direct the electrochemical

gradient of protons to the Fi moiety; the role of FI is to bind ADP and

Pi and t o use this electrochemical gradient to drive the synthesis of

ATP. The crystal structure of the FI domain from beef heart

mitochondria has been solved at 2.8 angstroms by Abrahams et al.

(160) and was found to be a fiattened sphere 80 angstroms high and

100 angstroms across. In al1 species in which this complex has been

studied so far, FI is composed of five subunits: a, P, y, 6, E and in

most species the subunit stoichiometry is a3 p g y 6 r. The three a and

p subunits are arranged alternately like segments of an orange

around a central a-helix which is 90 angstroms long and is formed

by the C-terminus of the y subunit. This helical structure protrudes

from the main body in a stem and most Iikely constitutes a part of

the "stalk" between the FI and Fo moiety (160). The a, P, and y

subunits are well conserved with the p displaying 70% similarity

from E. coli to humans. These $ subunits contain the three catalytic

sites and react with di- and triphosphates of purine and pyrimidine

nucleotide substrates. The structures of the three catalytic sites

were found to always differ, whereby each one is believed to pass

through a cycle of "open", "loose", and "tight" States (160). This is in

agreement with the prediction of an earlier, low resolution X-ray

structure, which showed that the FI domain is asymmetrical (161).

The a subunits, which are homologues of the P subunits, do not have

a catalytic role, but they bind ADP and ATP. Hence, there are six

nucleotide binding sites on the FI moiety, one on each a and P subunit. In addition t o the a, P , y, 6, and E subunits, there is an

inhibitor protein, IF,, that associates with FI at low protonmotive

force and inhibits ATP hydrolysis.

The subunit composition of the Fo portion varies among

species; the human one consists of a t least eight different types of

subunits: a, b,, ~~(stoichiornetry varies but does not exceed 12), d, e,

OSCP (oligomycin sensitivity conferring protein), F6 (=Factor 6), and

A6L. In S. cerevisiae, the ATP synthase consists of only three

subunits. In al1 species examined so far, subunit c, which together

with the fourth and fifth transmembrane helices of subunit 6 forms

the proton channel, is involved in proton conduction via a free

carboxyl group ( 7 8 ) . Factor 6 is required for FI binding to Fo, and

OSCP is involved in energy coupling between FI and Fo.

ATP synthesis on the surface of the F1 moiety has been shown

t o need little input of energy. The reaction step that actually

utilizes the energy from the electrochemical gradient is the binding

of substrates and the release of bound ATP from Fi (76. 77). Since the

ATPase complex can also work in reverse, catalyzing the hydrolysis

of ATP, it is important that some kind of regulation exists in order

t o prevent the hydrolysis of the newly synthesized ATP. In humans

this is achieved in two ways: 1) binding of the inhibitor protein IF1

to sites on the a and p subunits and 2 ) binding of ADP to sites on the

a and p subunits. Hence, by occupying these sites, IF1 and ADP

prevent the binding and subsequent hydrolysis of ATP. This model

suggests that under high protonmotive force, when IF, or ADP corne

in contact with the proton gradient, IF1 will be released from the

binding sites whereas ADP will be converted to ATP. Hence, in this

model the substrate-binding site will also be the inhibitory site (77).

Nucleo-mitochondrial in teractions

As previously mentioned, mitochondria are believed to have

evolved from an endosymbiotic relationship. Hence the eukaryotic

cell was assembled by a series of symbiotic events. This means

that nucleus, mitochondria, and chloroplasts were each derived from

a different phylogenetic lineage (1. 3). As a result, mitochondria

contain their own genome, but it encodes only a Iimited number of

proteins necessary for the biogenesis and function of this organelle.

The majority of proteins are encoded by nuclear DNA. Hence, for the

bc7 complex and the other complexes that are composed jointly of

nuclear and mitochondrial gene products, the nuclear encoded

subunits have to be targeted for import into mitochondria where

they are assembled with their partner subunits to form functional,

multisubunit enzyme complexes. For this to occur, there has to be a

coordination of the two separate genetic systems.

How do mitochondria and the nucleus communicate?

The answer to this question is far from understood, however

there are a number of levels at which regulation can occur. These

are transcription, translation, import into mitochondria, and

assembly. Saccharomyces cerevisiae is a facultative anaerobe that

can tailor the level of mitochondrial biogenesis to its specific needs

in response to its environment; under different conditions of growth

different metabolic pathways are activated ( '3) . Under conditions of

starvation, for example, the enzymes of the gluconeogenesis

pathway become operative. On the other hand, when yeast are grown

on media containing high levels of glucose, many respiration related

proteins suffer from glucose repression, that is the uptake systems

of sugars other than glucose are repressed (a phenomenon also

referred to as catabolite repression) (2. '5). Expression or repression

of certain genes is usually regulated a t the level of transcription

and is dependent on heme, oxygen, "actively respiring mitochondria",

compounds from intermediary metabolism, and other factors. For

example, glucose repression of mitochondrial biogenesis is in part

attributable t o a three- to sixfold decrease in the amount of

transcription of mitochondrial genes (79). Another example of

regulation a t the transcriptional level is the induction of alcohol

dehydrogenase II and other enzymes of the gluconeogenic pathway.

This induction does not occur in respiratory deficient mutants or in

wild type cells grown on minimal media. Conversely, under

conditions when yeast need oxidative metabolism, growth and

division of the cell are matched by an increase in mitochondrial

mass (13). Although yeast seem to "prefer" the anaerobic growth on

glucose, they cannot grow in the complete absence of oxygen since it

is needed for the synthesis of heme, egosterol, and unsaturated fatty

acids. A mutation resulting in a respiration-deficient phenotype or

addition of an inhibitor to the respiratory chain in combination with

depletion of ATP in the mitochondria is lethal to S. cerevisiae.

There are several types of lactate dehydrogenases, specific for

either L- or D- lactate, that can be synthesized depending on the

growth conditions of S. cerevisiae. On high concentrations of

glucose, whether yeast are grown aerobically or anaerobically, an

NAD-linked dehydrogenase is induced. This lactate dehydrogenase

reduces pyruvate t o lactate and thus regenerates NAD, thereby

keeping the NAD/NADH redox couple balanced (2. 80. 81). However,

this lactate dehydrogenase can also be induced a t low glucose

concentrations when the mitochondrial respiratory chain or

mitochondrial protein synthesis are inhibited. There are two other

types of lactate dehydrogenases that are synthesized when yeast are

grown aerobically on lactate: a D-lactate:cytochrome c

oxidoreductase and a L-lactate:cytochrome c oxidoreductase. These

enzymes catalyze the oxidation of lactate to pyruvate and the

electrons from lactate reduce cytochrome c of complex IV in the

mitochondrial respiratory chain. Thus growth on lactate, unlike

growth on ethanol or pyruvate, can often still occur in the presence

of antirnycin since the proton gradient established by cytochrome

oxidase generates enough ATP t o sustain slow growth (2). When

yeast are grown on galactose the majority of mitochondrial enzymes

are derepressed and ATP is derived from the respiratory chain.

The ex~ression of manv cornplex III subunits is dependent on thg

need for oxidative metabolism

Steady-state levels of the subunits of the bel complex and

cytochrome c of complex IV Vary in response to the need for

oxidative meta bolism. For cytochrome c, this response is primarily

governed by the rate of transcription (82-85). The nuclear gene

encoding cytochrome c, CYC 7 , has two upstream activation

sequences, UAS1 and UASZ. An activator protein called HAP1 binds

to UAS1 and increases RNA synthesis 300-fold under aerobic

conditions; this binding of HAP1 t o UAS1 is stimulated by the

presence of heme. Oxidative phosphorylation is therefore regulated

by the intracellular level of heme, which is logical, since the final

stages of heme synthesis occur in the rnitochondrion and are

dependent on the level of oxygen. On the other hand, CYC1 is

affected by glucose repression (86, 87). There is evidence that HAP1

control is also exerted on genes that encode proteins without hemes,

such as core protein 1 of complex 111 (44).

Transcription of the nuclear genes QCRG, QCR7, QCRB, QCR9,

and the genes encoding core protein 2, cytochrome c l , and the ISP are

al1 repressed by glucose (4% 44). Most of these genes contain the

consensus sequence element of UASZ, but it is not known whether

this is involved in the catabolite repression. The QCR9 gene might

also be regulated at the level of mRNA splicing. This gene has an

intron, and since introns are rare in yeast genes, they often serve a

function, such as the intron of the mitochondrial cytochrome b gene.

The intron of the QCR9 gene has some identical sequence elements to

the intron within the COX4 gene; these sequences might regulate the

coordinate expression of the bc, complex and the cytochrome oxidase

complex (44).

Coordination o f the expression of nuclear and mitochondrial

genes encoding subunits of the mitochondrial respiratory chain can

involve the direct regulation of expression of these genes, or the

regulation of genes whose products are involved in the synthesis of

individual mitochondrial products. Examples of the latter include

gene products involved in the S'-end processing and intron excision

of the pre-mRNA of cytochrome b and in its translation (83-85).

Another example is the CBP6 gene, a nuclear gene coding for a

protein that stimulates translation o f cytochrome b mRNA; by

suppressing this gene, synthesis of cytochrome b could be decreased

(88). Furthermore, cytochrome oxidase subunits 1 and 2 require

nuclear gene products for the processing and translation of their

transcripts ( 1 5 ) . An example of a nuclear encoded protein that

regulates mitochondrial function a t the level of assembly is

provided by the RCA 7 gene product. If this gene is mutated,

assembly of the ATPase subunits into a functional complex V is

prevented (89). As can be seen, there are rnany nuclear encoded

enzymes that are necessary for transcription of m t DNA's,

processing of precursor RNA's, and translation of mitochondrial

messages. In addition, RNA polymerase and almost al1 the proteins

that constitute the mitochondrial translational machinery are

encoded by nuclear DNA. An example of possible regulation by the

proton gradient in the IMS is given by the Qcr6 protein of the bc7

complex. In experiments where the QCRG gene was deleted, half of

the reduction sites of the dimeric bc7 complex were silenced.

Results of these studies suggested that under physiological

conditions when concentrations of ADP are low and ATP is abundant,

the increase in the positive charge surrounding subunit 6 may cause

it to regulate the complex so as to silence half of the reduction

sites and thereby downregulate the activity of the bel complex (46).

Do mitochondria exert reaulation on nuclear aene products?

All the above examples include regulation exerted by nuclear

genes on mitochondrial function and biogenesis. What about the

regulation of expression of nuclear genes by mitochondria? An

argument against the existence of such a regulation is that the

synthesis of PET products, nuclear genes required for the

morphogenesis o f respiratory competent mitochondria, is not

dependent on the presence of an intact mitochondrial genome (15).

Expression of many PET gene products is unaltered in p- and p o

strains, strains with large mitochondrial deletions or no mt DNA a t

all, respectively. Mitochondria of po cells can therefore be very

similar to wild type cells and can contain many functional enzyme

complexes.

Although not abundant, there is some non-specific evidence for

the regulation exerted by mitochondria on the nuclear genorne.

Experiments performed in N. massa suggested that treatment of

cells with chlorarnphenicol, an inhibitor of the mitochondrial protein

synthesis apparatus, increases the synthesis of some nuclear

encoded proteins that are part o f the mitochondrial transcription

and translation apparatus. This study may suggest that nit DNA

encodes a repressor protein that regulates nuclear genes (90). This

conclusion seems far-fetched and it is more probable that the

increase in synthesis of nuclear encoded proteins is the reponse t o

the lowered ATP levels in the cell. Another study ernployed

subtractive hybridization and indicated that some transcripts of the

nuclear genome Vary in abundance by a factor of five or more among

derepressed isochromosomal cells depending on whether they were

part of a p -, po, or mit- cell (91).

Pro tein import in to mitochondria

Localization of a protein into its correct compartment of the

cell is essential since mislocalization can lead to severe diseases.

Lysosomal storage disease, for example, in which degradative

enzymes are erroneously secreted into the bloodstream, results in

the accumulation of undegraded products in the lysosome. The

accumulation of glycosaminoglycans or glycolipids in the lysosome

is the cause for 1-cell disease (92). Another example of a disease

that can be caused by a targeting defect is primary hyperoxaluria.

This disease can sometimes be the result of erroneous targeting of

the peroxisomal enzyme L-a1anine:glyoxylate aminotransferase into

mitochondria.

As already mentioned earlier, only a srnall percentage of

mitochondrial proteins are encoded for by the mitochondrial gemme.

Hence, the vast majority of mitochondrial proteins are synthesized

on cytoplasmic ribosomes and must therefore be transported into

one of the four compartments of mitochondria, namely the OMM, IMM,

IMS, or matrix. Of the four final destinations in the mitochondrion,

import into the matrix is the best characterized. However, virtually

al1 mitochondrial precursors are initially imported by the same

machinery (Fig. 1-4; please refer t o this figure throughout this

section).

j m ~ o f t into the mitochondrial matrix

lmport into the matrix is the best characterized pathway. I t

constitutes the route into mitochondria for many precursors. Even

precursors with final destinations other than the matrix are often

imported into this cornpartment first, before being further

transported to their final destinations. The sequence of events is as

follows:

1. Precursors usually have an amino-terminal targeting

sequence (also referred to as signal-, leader-, transit-, or

presequence) that is recognized by cytosolic factors which

escort the protein to recepton in the OMM.

Fig. 1-4. lmport of proteins into the mitochondrial matrix, as adapted from Schatz (93). The precursor protein may bind to a cytosolic chaperone (MSF, mitochondrial import-stimulating factor, or hsp70) and then to the receptor (R) in the OMM which transports it to the protein import channel. Subsequently, the signal sequence of the precursor inserts itself into the protein import channel (which consists of TOM42, 8, and 6 in the OMM and TIM17, 23, and 6 in the IMM) and is pulled across the membrane by the mhsp70-GrpEp-TIM44 complex; translocation across the IMM requires an electrical potential across this membrane, AT. The mhsp70-GrpEp-TIM44 complex resides on the matrix side of the membrane and interacts loosely with the import channel in the IMM. Once in the matrix, the targeting sequence of the precursor is cleaved by the matrix processing peptidase (MPP). The precursor is most Iikely aided by the rnitochondrial chaperone hsp60-cpn10 complex in refolding and assembly in to a rnultimeric enzyme complex, if appropriate.

Precursor Protein

+++ y"'

ATP " "y&

IMM

2. Precunors bind proteinaceous receptors on the surface of

the OMM.

3. Receptors transport precursors t o translocation channels

which are opened by the signal sequence.

4. The precursor is translocated across the IMM by a protein

that is located on the matrix side of the membrane. Transport

across the IMM requires an electrical potential, acp, across the

membrane.

5. The protein folds on the matrix side of membrane with the

help of chaperones and its signal sequence is cleaved off.

Proteins that are part of a multimeric enzyme complex are

aided in their assembly by chaperones.

The taroetino seauence

Many, but not al1 mitochondrial proteins have an amino-

terminal targeting sequence that is cleaved on the trans side of the

membrane (94). Targeting sequences lack a consensus and up to 25%

of randomly generated peptides can function as mitochondrial signal

sequences (95). Nonetheless, there are some general features that

most matrix targeting signals have in common; they are 15-35

residues long, rich in hydroxylated and basic amino acids, and

capable of folding into an amphipathic a-helix (9% 94). Hence, signal

sequences display highly degenerate prirnary sequences, but their

common features result in a similar distribution of charged/polar

and hydrophobic residues. This common distribution of residues

results in the formation of an amphipathic helix which is essential

for the function o f signal sequences and renders them more

effective in targeting than randomly generated peptides.

Experiments in E. coli have shown that targeting sequences have two

functions; t o retard folding of the precursor, and t o recognize the

receptor on the membrane. Removal o f the signal sequence was

found to inhibit targeting by a factor of 100 to 1000 fold (96.97).

Some proteins of the IMS contain composite or bipartite signal

sequences. Cytochrome c l and the Rieske iron-sulfur protein of the

bc7 complex are two such examples. In these proteins the amino-

terminal part o f the leader sequence resembles the hydrophilic

matrix targeting sequence. The carboxyl half of the leader sequence

resembles a bacterial export sequence and is more hydrophobic than

its import counterpart (94).

osolic chaperones

Molecular chaperones are proteins that interact with non-

native conformations of other proteins such as mitochondrial

precursors. This interaction prevents aggregation o f the (often

newly synthesized) proteins until these have either folded correctly

or been transferred to a membrane receptor. There are two groups

of precursors, classified according t o their requirement for

extramitochondrial ATP (7. 9*v 99). The first group does not need ATP

for import into mitochondria and includes precursors of cytochrome

b r , holocytochrome c synthase, mitochondrial hsp60, and

dihydrofolate reductase fusion proteins. Hence, this group is

unlikely to interact with any ATP-requiring chaperones. The second

group of precursors is ATP-dependent and therefore is likely t o bind

to chaperones. This group includes alcohol dehydrogenase III,

cytochrome c l , the Flp subunit o f the ATPase complex V, and the

ADPIATP carrier. Two types of ATP-dependent cytosolic chaperones

have been identified t o date. MSF, or mitochondrial import-

stimulating factor, binds t o the signal sequence of a subset of

precursor proteins that have been released from the ribosome (7# 94).

Since this chaperone interacts specifically wi th mitochondrial

precursors, it most likely functions in escorting precursors to the

receptors in the OMM as well as in preventing aggregation. The

second type of ATP-requiring cytosolic chaperone is the cytosolic

heat-shock protein 70, hsp70 (98. 99). There are a t least two closely

related hsp701s in the cytosol of S. cerevisiae and they function

together with another protein called MAS5 or YDJ1 which stimulates

the release of precunor proteins from hsp70 (100). Hsp70 interacts

not only with mitochondrial precursors, but with a wide spectrurn of

non-native proteins targeted to different organelles and its function

is therefore not t o target but rather t o prevent aggregation and

misfolding (7. 94). Chaperones are important for maintaining proteins

in a partly unfolded state since most membrane systems can only

translocate proteins that are loosely folded (8. 101). Certain small

proteins that can spontaneously adopt a loose conformation, such as

the coat protein of the M l 3 phage, do not need the aid of chaperones

to maintain them in a transport competent state. However, most

large proteins and especially those with hydrophobic interiors

require the assistance of cytosolic factors (102).

Recentors in the OMM

Receptors in the OMM are mobile molecules that move within

the plane of the membrane and interact dynamically with the

translocation channel. Hence, one translocation channel could be

served by several different receptors whereby the function of the

receptors is most probably t o "trapu the precursor protein and to

transport it to a translocation channel. The receptors of the OMM are

integral membrane proteins with cytosolically exposed domains.

Depending on the type of receptor, it recognizes either the signal

sequence of a precursor protein or the precursor bound to a cytosolic

chaperone (94 ) . So far, four integral OMM proteins have been

identified in yeast that function as import receptors; these are

termed MASZO, MAS22, MAS37, and MAS70, consistent with their

molecular weights. MAS20 and MAS22 form a subcomplex that

contains a highly acidic region referred to as acid bristles. These

acid bristles are believed to bind the basic and amphipathic signal

sequence of precursor proteins in an ATP-independent manner.

MAS37 and MAS70 form a subcomplex that recognizes precursors

bound to the cytosolic chaperone MSF. Hence, this subcomplex most

likely recognizes the same features in the mature part of the protein

as the chaperone and this interaction also requires ATP. Due to the

dependency of MAS37-MAS70 on ATP, the group of precursors that is

preferentially imported by this subcomplex is prone to aggregation

and thus bound to cytosolic chaperones. As already mentioned above,

this group includes ADHIII, cytochrome c l , F$, and the ADP/ATP

carrier (7, 9, 103, 104).

It is not known whether the two subcomplexes function

separately or together and there are currently two different rnodels

regarding the association of the two receptor subcomplexes MASZO-

MAS22, and MAS37-MAS70. The first model suggests that they act

independently; hence for a given precursor the majority of that

precursor will bind preferentially to one of the subcomplexes. The

second model States that the two subcomplexes function in a joint

fashion whereby al1 precursors use the same pathway and several of

the receptor subunits are necessary for the recognition of one

precursor. Examples to substantiate the second model include the

irnport of ADHlll and FIP. Studies with these precursors found that

they are bound to both subcomplexes; the signal sequence is bound to

the MAS20-MAS22 subcomplex and the mature part of the protein is

bound to MAS37-MAS70. Another example is given by the ADP/ATP

carrier. Although this protein does not have an amino-terminal

signal sequence, but rather contains the targeting information

within i ts mature region, it was also found to bind to both receptor

subcomplexes. Since the MAS20-MAS22 subcomplex binds

precursors in an ATP-independent fashion, only precursors that are

imported in a manner that does not require extramitochondrial ATP

interact with this subcomplex. The two-hybrid systern was used to

show that the two subcomplexes associate loosely with one another

(los) and that the cytosolic domains of MASZO and MAS70 interact (7).

Deleting the genes encoding the receptors prevents transport

of different precurson to varying extents. Hence, a given precursor

may be presented to the membrane system by different routes

although it may have a preference for a certain receptor or receptor

subcomplex. Deleting the genes encoding receptor subunits MAS20,

MAS37, and MAS70, respectively, results in strains that are still

viable; however, deleting the genes encoding both MAS37 and MAS70

is lethal. MAS22 is the only subunit that is essential for viability

since deletion of this subunit alone is lethal. This finding may

reflect the fact that the acidic domain may bind the presequence,

and the fact that MAS22 spans the OMM and thus constitutes part of

the transport system across the OMM (7,941.

The translocation channel and the mitochondrial chaoerones

W hen conside ring the exposed charges of the mitochondrial

signal sequence and the hydrophilic side chahs of the partially

unfolded polypeptide, one may wonder how the protein can cross the

hydrophobic phospholipid bilayer. The answer is that the

translocating protein passes through a hydrophilic, hetero-

oligomeric transmembrane channel that is composed of integral

membrane proteins. How exactly this translocation takes place is

not understood, however, the channel is opened across the membrane

by the signal sequence. Lateral movement through the membrane is

mediated by the stop-transfer of signal-anchor sequences. lmport

channels span both the IMM and OMM and they are situated at "contact

sites" where the two membranes are in close contact with each

other. The channel in the OMM consists of approximately three

subunits; TOM42 (formerly called ISP42), an essential protein, as

well as TOM6 and TOM8. The channel in the IMM consists of TIM17

(formerly MIM17), TIM23, TIM6 (formerly MASG), and TIM44

(formerly MIM44 or ISP45). TIM44 is bound to mhsp70 on the trans

side of the membrane, and the latter in turn is bound to i ts CO-

chaperone GrpEp. The mhsp70-GrpEp-TIM44 complex interacts

loosely with the import channel in the IMM and operates as a

"translocation motor" that binds the emerging signal sequence and

"pulls" the precursor across the membranes. Translocation across

the IMM membrane requires intramitochondrial ATP and is dependent

on the electrochemical potential for most precursors (1 06-1 0).

Precursors that have been translocated into the matrix are

partially unfolded and have to be refolded in order to be able t o

assume their biological functions. Some small, monomeric proteins

have the ability to fold rapidly by themselves. However, others

require the help of mitochondrial chaperones ( y l l p 112). Hsp60 and

cpnl0 forrn a complex that operates in an ATP-dependent manner to

refold translocated proteins and assemble them into multimeric

enzyme complexes where appropriate. In addition, mhsp70 also aids

in the folding of proteins as well as in binding misfolded proteins

and delive ring them for degradation (1 3-1 6).

lm 1 The import pathway into the IMS, which can be reached by

several possible routes, is not well understood. Cytochrome c of the

cytochrome oxidase complex, for example, spontaneously inserts

itself into the OMM (l17) and is then pulled across the OMM by the

enzyme cytochrome c heme lyase which catalyzes the covalent

attachment of heme to cytochrome c in the IMS (6. 117). This protein

does not require the aid o f chaperones or receptors and is therefore

imported in a manner that requires neither ATP nor a membrane

potential.

A different route is taken by the precursors of cytochrome cl

and cytochrome bt which contain bipartite signal sequences, cieaved

in two steps (118, 119). There are currently two views concerning

how these proteins reach their final destination. In the first one, a

stop-transfer pathway, the proteins are transported across the OMM

and then become anchored t o the outer face of the IMM by their

signal sequence which is subsequently cleaved off. After rernoval of

the amino-terminal part of the leader sequence the proteins remain

bound to the outer face o f the IMM or are released into the IMS. The

carboxy half of the signal sequence thus functions as a sorting

signal that prevents transport of the intermediates into the matrix

and it is cleaved off by a peptidase in the IMS (118. lzo). The second

pathway is referred t o as the conservative route in which the

precursors are first irnported into the matrix where the signal

sequence is cleaved for the first time and where they interact with

hsp60 (1219 122) and mhsp70 (123). In this pathway, the carboxy-

terminal end of the signal sequence functions as an export signal

that resembles that o f bacteria and i t targets the intermediates

from the matrix across the IMM into the IMS. Additionai proteins

that are conservativeiy sorted include the ISP of the bcl cornpiex and

the ATPase subunit 9.

Cytochrome c heme lyase (CCHL) is sorted in yet a different

manner from cytochromes cl and b2. This protein does not contain a

presequence and is translocated selectively across the OMM by using

the import channel (124). CCHL is believed to be loosely folded after

synthesis since it does not require ATP or a membrane potential for

import and therefore does not interact with cytosolic chaperones.

lm-wrt into the IMM

There are two known routes for a protein if its destination is

the IMM, the "direct" route and the "detour" route (101). In the direct

route the protein is transported across the OMM and directly

inserted into the IMM from the IMS side. An example for this import

pathway is the ADP/ATP carrier (125). The import of the ADP/ATP

carrier requires extramitochondrial ATP, hence it probably interacts

with a cytosolic chaperone. lmport across the OMM is dependent on a

proteinaceous component on the surface of this membrane, most

probably a receptor, and the protein import channel. In addition,

insertion into the IMM requires a membrane potential. There is no

requirement for matrix ATP, an indication that the protein does not

pass through the matrix to reach its final destination in the IMM.

The detour route resembles the import pathway into the matrix

or the conservative pathway taken by some proteins of the IMS. In

this pathway, proteins are imported into the matrix first where the

matrix signal sequence is cleaved off; this import into the matrix

occurs in a manner that is dependent on receptor interaction, extra-

and intramitochondrial ATP and the membrane potential. Proteins

are then assembled with their partner subunits, if appropriate, and

inserted into the IMM from the matrix side. Proteins that follow

this indirect route include cytochrome oxidase subunit IV in yeast,

ATPase subunit 9, and the Rieske ISP of the bel complex (101, 1269

127).

jmwn into the OMM

lmport into the OMM is the simplest pathway, however the

mechanism of import is not well understood. Transport into the OMM

is receptor dependent for most proteins and occurs by direct

insertion of the protein into the membrane. Thus import into the OMM

requires extra-, but not intramitochondrial ATP; neither a membrane

potential nor intramitochondrial components o f the import

machinery are required (101. 128). It is therefore not understood how

transport into the OMM is energized. There is some preliminary

evidence t o suggest that proteins are driven across the membrane by

a two-step sequential binding. In the first step the presequence

associates with a receptor on the surface of the OMM and in the

second step, which drives the translocation of the protein across the

membrane, the precursor binds on the trans side of the OMM (lin 129).

Details about the signals that encode the pathway into the OMM are

unknown.

Objectives of the research

Abundant research has been performed on those subunits of

complex III that contain catalytic centers. On the other hand,

knowledge about the functions of the supernumerary subunits is still

limited. The Qcr7 protein was chosen as the focus of this study

based on the knowledge that subunit 7 is crucial for the functioning

of complex 111 (33). In addition, study of this subunit was appealing

because it constitutes the homolog to the fourth polypeptide of

prokaryotic bci complexes which consist of only four subunits: the

three catalytic subunits cytochrome b, cytochrome c or f , the Rieske

ISP, and the homolog t o the Qcr7 protein in yeast.

In this study I focused on the role of the amino-terminal

region of subunit 7. The N-terminus of subunit 7 is thought to face

the matrix side of the IMM in yeast (1301, and in the homologous

subunit of beef heart mitochondria (131). Because proteolysis of the

N-terminal seven amino acids of the beef heart mitochondrial

counterpart leads to a decreased H'/e- ratio, Cocco e t al . (131)

suggested that this subunit is involved in proton uptake from the

matrix w i th subsequent transfer of these protons t o the

hypothetical ubiquinone binding pocket a t center N. Since

proteolysis also resulted in a small amount of cleavage from the

Rieske ISP, it cannot be ruled out that the ISP is in fact responsible

for the decreased H+/e- ratio. Nevertheless, based on the available

information of subunit 7, 1 decided to investigate the function of the

Qcr7 protein with an emphasis on the amino-terminal region.

CHAPTER 2

Inactivation of the QCR7 Gene

A synopsis of the work presented in this dissertation has been accepted

for publication in The Journal of Biologieal Chemistry.

CHAPTER 2

Inactivation of the QCR7 Gene

2.1 . INTRODUCTION

The yeast Saccharomyces cerevisiae is an ideal eukaryotic

microorganism for the study o f biochemical pathways and is

frequently used as a model system for investigating the function of

proteins. Yeast have greater genetic complexity than bacteria.

However, they share many of the technical advantages that have

permitted rapid progress in the understanding of the molecular

genetics of prokaryotes. Some o f these advantages include the

availability of strains with multiple auxotrophic markers, rapid

growth, a well-def ined genetic system, a highly versatile DNA

transformation system, and the ability of the organism to exist both

in the diploid and haploid state. In addition, replica plating in yeast

can be used as a quick preliminary identification of mutants ( i 3 2 ) .

The ability of wild type yeast to grow aerobically and anaerobically

allows for the rapid preliminary identification of mutants defective

in aerobic growth and makes this organism very attractive for the

study of mitochondrial assembly and function.

Many genes have been inactivated in yeast and the resulting

phenotypes have contributed significantly toward understanding the

functions of proteins in vivo. All the genes encoding nuclear

subunits of ubiquinol-cytochrome c oxidoreductase (complex III or

bc, complex), including the QCR7 gene for subunit 7, have been

In this study I attempted to determine the precise function of

subunit 7 (the Qcr7 protein) by the expression of a series of mutated

Qcr7 proteins in the yeast S. cerevisiae. To accomplish this, I had to

first create a strain in which the chromosomal copy of the QCR7

gene was inactivated so that a Qcr7 protein is not synthesized. This

chapter describes the disruption of the QCR7 gene to create the

qcr7A:LEUZ allele-containing respiration deficient strain, YSM-

qcr7A, which was used for the expression studies outlined in the

following chapten.

2.2. MATERIALS AND METHODS

Ma terials

Saccharomyces cerevisiae strain W303-1 B (MA Ta, ade2- 7,

his3-71, 15, ura3-7, leu2-3, 712, trp7-7, can7-700), which was

used as the parent strain throughout this study, and the pJJ250

plasmid were kind gifts from Dr. Jim Friesen's laboratory (Hospital

for Sick Children, Toronto). The rho- strain (MATa, ural [rho-]

[ C X 1 R ES 1401) was purchased from ATCC (strain number 42209;

donated by Dr. L. A. Grivell, University of Amsterdam). Escherichia

coli strain JMED3 was obtained from Promega (Madison, WI).

Restriction enzymes, the T7 sequencing kit, and NlCK gel filtration

columns were purchased from Pharmacia (Montreal, PQ). All

materials for the preparation of yeast and bacterial media were

obtained frorn Difco (Toronto, ON) or ICN (Costa Mesa, CA). The pCR

II vector was purchased from lnvitrogen (San Diego, CA). All

remaining chernicals were purchased from Sigma (St. Louis, MO) or

BDH (Toronto, ON). Alkaline phosphatase-conjugated AffiniPure

rabbit anti-chicken IgY (IgG) (H+L) was obtained from Jackson

lmmuno Research Laboratories, Inc. (West Grove, PA). Random

primed labeling kits were from Boehringer Mannheim (Mannheim,

Germany), and Hybond-N' nylon membranes and radionucleotides

were from Amersham (Arlington Heights, IL). BiomaxTM MS x-ray

film was purchased from Kodak (Rochester, NY). Nitrocellulose

membranes were purchased from Mandel Scientific Company Ltd.

Serocluster vinyl plates used for ELISA assays were obtained from

Costar (Cambridge, MA). NBT (p-nitro blue tetrazolium chloride) and

BClP (5-bromo-4-chloro-3-indolyl phosphate-toluidine) were

purchased from BioRad.

2.2.1. Disruption of the chromosomal copy of the QCR7 gene

The QCR7 gene was amplified from genomic yeast DNA (136)

using primers corresponding to sequences upstream (5'-

c t g t a a t t a a a c g t t c c a g a a a g - 3 ' ) and downstream (5 ' -

cgggttgtgtgttcgtggtga-3') of the coding sequence. Amplification

was perforrned for 35 cycles with a denaturation time of 1 min a t

94<, an annealing time of 1 min a t 65oC, and a 1 min extension a t

720C. The PCR product was subcloned into the pCR II cloning vector

by using the 3' adenylic acid overhang created by Taq DNA

polymerase and giving pCR II-QCR7. Eight clones were used to

confirm the sequence and orientation of the insert by dideoxy chain

termination sequencing.

A Pvull fragment (2300 bp) containing the LEU2 gene was

purified from plasmid pJJ250 and blunt-end ligated into the Hincll

site of pCR II-QCR7 to give pCR Il-Aqcr7 in which the qcr7 gene is

disrupted by the LEU2-containing fragment. The vector pCR Il-dqcr7

was digested with Bgll and Apal to liberate the DNA fragment

containing the disrupted qcr7 gene (Fig. 2-1 ) . Ten micrograms of

digested DNA was used to transform yeast strain W303-1B (137).

Appropriate integrative transformation was confirmed by phenotypic

analyses and by Southern blot analysis of genomic DNA from Leu+

transformants.

1 acr7, LEM qcr7 C

KR II vector

a qcr7codng region

vector pJJ250

LEU-containing fragment

Fig. 2-1. DNA fragment used for disruption o f the chromosomaI QCR7 gene. The triangles represent the direction of transcription.

2.2.2. Confirming inactivation of the QCR7 gene

Selection of diploids

Diploids were obtained by inoculating YPD medium

simultaneously with cells from a rho- strain and cells from a Leu+

transformant containing the putative qcr7 gene disruption. The

culture was grown overnight, cells were washed twice with water,

diluted, and diploids were selected on SD medium lacking leucine and

containing glycerol and ethanol as the sole carbon sources.

Northern and Southern blotting

Total yeast RNA was prepared by growing a 25 mL culture

overnight, harvesting the cells and resuspending the pellet in 2 mL

of AE buffer (50 mM NaOAc, pH 4.8; 10 mM EDTA). The suspension

was vortexed after the addition of 200 pL of 10% SDS, and again

after the addition of 450 pL of phenol saturated with AE buffer. The

suspension was vortexed three times during a five minute incubation

a t 650C. The mixture was extracted with phenol-chloroform (1 : l )

and RNA was precipitated from the aequeous phase with 2.5 volumes

of ethanol. The pellet was washed with 70% ethanol, dried, and

resuspended in TE buffer (pH 7.0). For the Northern analysis, 30 pg

of RNA was precipitated with 2.5 volumes of ethanol-0.1 volumes of

5 M LiCI. The RNA pellet was dissolved in 5 IL of 25 mM EDTA-0.1 %

SDS. A DNA fragment encompassing the entire QCR7 coding region

(48) was amplified by PCR and subsequently radiolabeled with [a-

WPI~CTP using a random primed labeling kit; unincorporated

nucleotides were removed from the labeled probe by gel filtration

through a NlCK column. Northern blotting was performed as

described by Fourney e t al. (1 38). Following hybridization, the

membranes were washed twice a t 650C for 30 min in 2 x SSPE

(20xSSPE: 3 M NaCI, 0.2 M NaH,PO., 0.02 M EDTA, adjusted to pH 7.4

with Na0H)-0.1% SDS and subsequently exposed to x-ray film.

Ten micrograms of yeast genomic DNA, isolated as described

by Strathern and Higgins ( 136 ) , was digested with appropriate

restriction endonucleases and electrophoresed through a 0.6%

agarose gel. The DNA was denatured in 1 M NaOH for one hour and

subsequently transferred in this solution to a Hybond-Ne nylon

membrane as outlined by Sambrook e t al. (139). Prehybridization was

performed a t 420C for a minimum of four hours in 40% formamide-

1% SDS-Sx SSPE-0.5% skim milk powder and 250 rng/mL of

denatured, sonicated salmon sperm DNA. A DNA fragment containing

the QCR7 coding region was radiolabeled with [a-IPI~CTP as

described previously. Hybridization was carried out a t 42oC

overnight in a solution containing 40% formamide-1 % SDS-5x SSPE-

0.5% skim milk powder-10% dextran sulfate. Following

hybridization, membranes were washed twice a t 650C for 30 min in

2x SSPE-O. 1 % SDS and were exposed to x-ray film.

Isolation of yeast mitochondria

Cultures were grown for two days in SD media containing

different carbon sources. Mitochondria were isolated essentially as

outlined by Guthrie and Fink (140) with the substitution of breaking

buffer (0.6 M sucrose, 20 mM HEPES-KOH, pH 6.5, 0.1% BSA, 1 mM

PMSF) for mitochondrial isolation buffer. In short, cells were

pelleted, washed with water, and then prepared for cell wall

digestion by incubation a t 30C in 0.1 M Tris-SOI (pH 9.4)-10 mM DTT

at a concentration of (0.5 g cell wet weight)/mL. After this

preliminary incubation, cells were washed with 1.2 M sorbitol and

incubated again at 30.C for cell wall digestion in spheroplasting

buffer (1.2 M sorbitol, 20 mM KPi, pH 7.4) containing zymolyase

(Img/g cell wet weight) and PMSF ( 1 mM). Spheroplasts were

disrupted by homogenization with a Dounce steel homogenizer in

breaking buffer (0.6 M sucrose, 20 mM HEPES-KOH, pH 6.5, 0.1% BSA,

1 mM PMSF). Following disruption of the spheroplasts, mitochondria

were isolated by differential centrifugation (two rounds of 3,000~9

followed by 10,000xg whereby mitochondria are pelleted a t the high

speed spin). The purity of the mitochondria was determined by

measuring the specific activity of complex IV.

Western analysis

Following SDS-PAGE using gels containing 16% polyacrylamide,

proteins were transferred to nitrocellulose membranes for two

hours a t 5 5 volts a t 40C in l x running buffer (0.025 M Tris, 0.1 9 M

glycine) containing 0.1 % SDS and 20% methanol. Biots were

subsequently blocked for one hour with Blotto (10 mM Tris-CI, pH

7.5; 150 mM NaCI; 0.05% Tween 20) containing 2% gelatin, and then

incubated overnight with primary antibody in Blotto containing 1 %

gelatin. Membranes were washed 4x30 min in Blotto and then

incubated in Blotto containing 1% gelatin for one to two hours with a

rabbit anti-chicken antibody coupled t o alkaline phosphatase.

Membranes were washed 4x1 5 min in Blotto and proteins were

visualized using NBT and BClP as substrates in 0.1 M NaHC0,-1 mM

MgCl,.

2.2.3. Raising an antibody against the Qcr7 protein in

chicken

The following peptide from the carboxy-terminus of the Qcr7

protein was chosen as antigen and synthesized by the Alberta

Peptide Institute:

keyhole limpet hemocyanin-AAKEKDELDN IEVSK-COOH

Isolation of anti-Qcr7 antibody from rabbit and chicken

The above peptide which is conjugated t o keyhole limpet

hernocyanin a t the amino-terminus, was used to raise an antibody in

rabbit (performed by the Centers of Excellence, Canada) and in

chicken. To raise the antibody in chicken, 250 pg of the peptide was

solubilized in 500 p l PBS, mixed with an equal volume of Freund's

Complete adjuvant and injected into the chest muscle of a chicken.

Booster shots containing 250 pg of peptide solubilized in 500 pL PBS

and mixed with an equal volume of Freund's lncomplete adjuvant

were given one and two weeks after the initial injection. Antibodies

were purified from egg yolks starting two weeks after the second

booster injection.

Three egg yolks (50 mL) were aspirated through a syringe

(without needle) and diluted in three volumes of 0.1 M sodium

phosphate buffer, pH 7.6. One volume of a PEG8000 solution (0.1 75 g

PEG8000/mL of 0.1 M sodium phosphate buffer) was added. After 10

min the mixture was centrifuged a t 5000xg for 25 min at RT. The

resultant supernatant was filtered through 3 mm filter paper and

protein was precipitated by the adding PEG8000 (0.085 g/mL) and

incubating for 10 min a t RT. This suspension was centrifuged a t

5000xg for 25 min at RT. The pellet was dissolved in 2.5 volumes of

0.1 M phosphate buffer and proteins were precipitated by adding

PEG8000 (0.1 2 g/mL) and incubating for 10 min at RT. The

precipitate was recovered by centrifugation at 5000xg for 25 min at

RT and the pellet was dissolved in 0.25 volumes of 0.1 M phosphate

buffer and cooled to OoC on ice. After the addition of an equal

volume of cold 50% ethanol, the mixture was left on ice for

approximately four hours and then centrifuged a t 10,000xg for 25

min a t 4oC. The final pellet was dissolved in 0.25 volumes of 0.1 M

phosphate buffer and dialyzed overnight at 40C against 0.1 M

phosphate-0.1 5 M NaCl (pH 7.6). After the addition of 0.02% NaN3 as

preservative, the antibody-containing fraction was aliquoted and

stored a t -700C. The immune reactivity was tested by performing an

enzyme-linked immunoadsorbent assay (ELISA procedure was

devised by the Alberta Peptide lnstitute a t the Department of

Biochemistry, University of Alberta, Edmonton, Alberta).

2.3. RESULTS

Disruption of the QCR7 gene

As a first step in the study of mutant Qcr7 proteins, I

constructed a yeast strain in which the chromosomal QCR7 gene was

inactivated. The chromosomal QCR7 gene was replaced by

integrative transformation of a DNA fragment containing the qcr7

gene interrupted by the LEU2 gene (Fig. 2-1). lntegrative

transformation in yeast generally occurs by homologous

recombination, hence Leu' transformants could result not only from

replacement of the QCR7 gene by the qcr7:LEUZ sequence, but also by

recombination of the transforming DNA at the chromosomal leu2

gene; in addition, Leu+ transformants could result from random

recombination elsewhere in the genome. To eliminate the seléction

of a strain in which the qcr7:LEUZ fragment has recombined a t a site

other than the genomic QCR7 locus, I first carried out a phenotypic

analysis of the Leu+ transformants.

The QCR7 gene is required for respiration-dependent growth,

that is growth on non-fermentable carbon sources (33) . 1 therefore

patched Leu' transformants ont0 medium containing glycerol and

ethanol as carbon sources. Transformants that failed to grow on

this medium were pet - and therefore candidates for containing the

qcr7 gene disruption. However, mutations in the mitochondrial

genome can also lead to a pet- phenotype, hence I had to ascertain

that the respiration-deficient Leu+ transformants had a wild type

mitochondrial genome. This was performed by mating the Leu4

transforrnants with a rho- strain (a strain which contains large

deletions in its mt DNA) and testing the resulting diploids for their

ability to grow on ethanoVglycerol medium. Diploids that were able

to grow on this medium had to contain an intact mitochondrial

genome.

Finally Leu+, respiration-deficient transformants that

contained intact mitochondrial DNA were subjected to Southern blot

analysis to confirm that the QCR7 gene had been replaced by the

qcr7A:LEUP allele. A blot of genomic DNA was probed with an [a-

"PIdCTP-labeled DNA fragment encompassing the QCR7 coding region.

The hybridization pattern of DNA that had been digested with Hindlll

confirmed that a 2600 bp fragment containing a Hindlll site had

replaced the chromosomal QCR7 gene. Only one fragment containing

the uninterrupted QCR7 gene is seen in the lane containing DNA from

the parental strain, W303-1 B (Fig. 2-2, panel B, lane 1). This is as

expected since the QCR7 coding region does not have a Hindlll

recognition site (Fig. 2-2, panel A). On the other hand, in the lane

containing DNA from the mutant strain the large fragment is

replaced by two smaller fragments since the coding region of the

qcr7 gene was interrupted by the LEU2-containing fragment which

introduced a new Hindlll site (Fig. 2-2, panel B, lane 2). In summary,

the phenotypic and Southern analyses confirmed that I had obtained a

strain in which the chromosomal QCR7 gene had been replaced by the

qcr7d:LEUZ allele, thus creating the new strain YSM-qcr7A.

Fig. 2 - 2 . Southern blot analysis of the parental strain W303-1 B and strain YSM-qcr7~ with the qcf7d:LEU2 allele. Yeast genomic DNA (1 0 pg) was digested with Hindlll (refer to panel A) and electrophoresed through a 0.6% agarose gel. The DNA was denatured in 1 M NaOH and transferred to a Hybond-N* nylon membrane, followed by fixation of the DNA to the membrane by treatment with ultraviolet light (254 nm) for 10 min. Hybridization was carried out a t 420C with a fragment encompassing the QCR7 coding region that had been labeled with [a-"PIdCTP by a random priming method. Panel B: Lane 1; DNA from the parental strain, W303-1 B; lane 2; DNA from the strain containing the qcr7A:LEUP allele, YSM-qcr7A.

Hind II

Qm7 1 . 4 wiid type lows

Hin d l Il 2575 bp Hindi I I

0 no~codng regions iqstiearnanddownstrearn of chrmasomal QCR7gene

0 QCR7obding region

vector pJJ250

LEU'-containing fragment

Confirmation of the qcr7 gene inactivation

The DNA construct (Fig. 2-1) used to disrupt the QCR? gene

included the entire coding region of this gene. Hence, it is

conceivable that in the strain YSM-qcr7~ a fusion transcript

consisting of the 5' end of the qcr7 gene and the LEU2 gene is

synthesized which results in a fusion protein. Such a protein might

have a dominant negative effect. To rule out the presence of such a

fusion transcript, Northern blotting was performed. From Fig. 2-3 it

is evident that no such transcript is present in the lane containing

RNA from strain YSM-qcr7A (lane l ) , whereas a transcript of

expected size (approximately 680 bases) is present in the wild type

parental strain (lane 2). This indicates that a stable fusion

transcript is not synthesized and further substantiates the QCR7

gene inactivation.

An antibody was raised in chickens t o a carboxy-terminal

peptide of the Qcr7 protein. The antibody was purified from egg

yolks and the titer was compared t o that of pre-immune egg yolks by

means of an ELISA. The preimmune egg yolks did not react with the

peptide (the peptide being the same one that was used to raise the

antibody) used in the ELISA, whereas a substantial reaction of the

post-immune egg yolks could be seen with the peptide. The antibody

was authenticated by immunoblotting of SDS-PAGE fractionated

mitochondrial proteins (Fig. 2-4). A band o f expected size

corresponding to the 14.5 kDa Qcr7 protein is seen in the wild type

parental strain (lane 2). As anticipated, a Qcr7 protein is not

present in strain YSM-qcr7~ (lane l ) , which confirms that the

protein recognized &y the antibody is the Qcr7 protein rather than

another protein of similar size.

Fig. 2-3. Northern blot analysis of RNA isolated from the parental strain W 3 0 3 - 1 B and YSM-qcr7~. Northern blot of total RNA (30 pg) isolated from the parental strain W303-1B (lane 2) and strain YSM-qcr7a (lane 1) . Total RNA was separated on a 1% agarose gel containing 1.8% formamide and subsequently transferred to a nylon membrane. The DNA probe consisted of the QCR7 coding region which was radiolabeled with [a-"PIdCTP by using a random prirned labeling kit.

Fig. 2-4. Western blot analysis of mitochondrial protein from YSM-qcr7a and the parental strain W 3 0 3 - 1 B. Mitochondrial proteins (1 00pg) were dissolved in SDS-PAGE buffer containing D7T and heated for three minutes at 950C. Samples were run on a 16% polyacrylamide gel and then transferred to a nitrocellulose membrane. Blots were probed with a polyclonal antibody (dilution 1 :100) raised against a c-terminal peptide of the 1 4 kDa subunit. Lane 1 : protein from YSM-qcr7~; lane 2: protein from the wild type parental strain W303-1 B.

2.4. DISCUSSION

This chapter describes the inactivation of the QCR7 gene by

replacement of the functional chromosomal copy with a version of

the gene that was disrupted by the LEU2 marker. Since the qcr7 gene

was disrupted rather than replaced, I had to ensure that a fusion

protein was not synthesized. Northern blot analysis (Fig. 2-3)

demonstrated that a stable transcript or fusion transcript is not

synthesized. Western blot analysis indicates that a fusion protein

which contains an in frame fused Qcr7 protein C-terminus is not

synthesized either. Whether or not a fusion protein is synthesized,

that contains an out of frame fused C-terminal end of the Qcr7

protein, cannot be determined as the antibody epitope is located in

the C-terminus. However, it is extremely unlikely that such a fusion

protein would be functional and/or stable. Hence, the gene encoding

subunit 7 of the bci complex has successfully inactivated and the

newly created strain, YSM-qcr7A, which contains the qcf7A:L EU2

allele, was used for further analysis of the Qcr7 protein by site-

directed mutagenesis of plasmid-borne qcr7 genes.

Since recombination and gene conversion could occur between

the chromosomal gene and a plasmid-borne version of the gene, the

mutant strain was always propagated in a selective manner (lacking

leucine) when transformed with a version of the qcr7 gene on an

expression plasmid. This ensured that the chromosomal gene was

qcr7A:LEUZ. Reversion of YSM-qcr7~ back to wild type has not been

found to be a cause for concern.

CHAPTER 3

Mutagenesis of the QCR7 Gene

CHAPTER 3

Mutagenesis of the QCR7 Gene

3.1 . INTRODUCTION

With the QCR7 gene inactivation verified it was now possible

to proceed with in vitro mutagenesis of the gene and express the

encoded mutant Qcr7 proteins in the newly created strain, YSM-

qcr7A. This study focused on the amino-terminus of the Qcr7 protein

and three functions were investigated. The first was based on

studies performed on the bc, complex of beef heart mitochondria in

which the purified complex was solubilized and subjected t o

protease digestion. Proteolysis resulted in partial cleavage of some

or al1 of the following subunits, depending on the experimental

conditions: core protein 2, the Rieske iron-sulfur protein, the 6.4

kDa subunit, the 9.2 kDa subunit, and 7 to 11 residues from the N-

terminus of the 13.4 kDa subunit (the homologue of the yeast 14.5

kDa Qcr7 protein). When complex III was reconstituted into

phospholipid vesicles after proteolysis and tested for redox and

protonmotive activities, it was found that there was a significant

decrease in the H'/e- ratio. This decrease in the H+/e- ratio was

still present under conditions where the 13.4 kDa subunit had been

cleaved but, of the other subunits, only marginal cleavage of the

iron-sulfur protein had occurred. The authors concluded from this

observation that the amino-terminus of the 13.4 kDa subunit is

involved in binding protons from the matrix phase and in promoting

conduction of these protons t o the ubiquinone-binding pocket (131.

741). It was furthermore concluded that the amino-terminus of the

13.4 kDa subunit protrudes into the inner space of the phospholipid

vesicles since there was no proteolytic cleavage of this subunit

under conditions where the complex was inserted into phospholipid

vesicles prior t o digestion. This finding correlates with the

predicted topology of the 13.4 kDa subunit in vivo where it is

believed to be a monotopic protein that is bound t o the surface of

the IMM on the matrix side where it presumably interacts with the

membrane and other subunits of complex III (Fig. 3-1 ) (23. 65).

Similar to its beef heart rnitochondrial homologue, the N-terminus

of the yeast Qcr7 protein is also believed to be located at the matrix

side (66,130, 142).

The second putative role for the Qcr7 protein that I

investigated dealt with mitochondrial protein targeting and import.

The rnechanisms for mitochondrial targeting of the Qcr7 protein are

unknown. Of the nine nuclear encoded subunits of the ber complex,

five have been shown t o contain cleavable mitochondrial signal

sequences (2. 36-39, 43) of varying lengths: core proteins 1 and 2 have

cleavable signal sequences 1 7 and 1 6 residues long, respectively;

cytochrome cl and the ISP have bipartite signal sequences of 61 and

30 amino acids, respectively; subunit 6 has a presequence of 25

amino acids. Subunits 7, 8, and 9 only undergo cleavage of the

initial methionine and this is presumably also the case for subunit

10. Although the mechanisms of import o f several of the

components of complex III in yeast have been studied extensively,

I M S

IMM

Fig. 3-1. Helical wheelplots and folding pattern of the 13.4 kDa subunit of beef heart mitochondrial complex III; as predicted by Link e t al. (23). Hydrophilic regions are located in the matrix while helices are predicted to be located in the IMM. The amphipathic helix a t the immediate N-terminus is predicted to be located on the matrix side of the IMM.

nothing is known about how subunits 7, 8, and 9 are imported into

mitochondria.

Core protein 2 follows the general energy-dependent import

pathway into the matrix where its signal sequence is cleaved off (2).

It is not known how core protein 1 is imported, however, it most

probably follows the same pathway since it also contains a

cleavable signal sequence and its final destination is in the rnatrix

with a small fraction of the protein bound to the IMM. The ISP is

irnported into the matrix in a receptor and membrane potential-

dependent manner where its signal sequence is cleaved for the first

time. The protein is then exported t o the outer face of the IMM in an

energy-dependent manner and cleaved by a protease for the second

time (2. 42). There are currently two theories on the import pathway

of cytochrome c l : In the first one, a stop-transfer pathway, the

precursor is transported across the OMM and then becomes anchored

to the outer face of the IMM by its signal sequence which is

subsequently cleaved. After removal of the amino-terminal part of

the leader sequence the protein remains bound to the outer face of

the IMM or is released into the IMS. The carboxy half of the leader

sequence thus functions as a sorting signal in this pathway and

prevents passage of the intermediate into the matrix. Cytochrome ci

is cleaved for the second time by a peptidase in the IMS (118). The

second pathway is referred to as the conservative route and the

precursor is first imported into the matrix according t o the general

pathway where it interacts with hsp60 ( i l s ) and mhsp70 ( t z i ) . In

this pathway the carboxy terminal end of the signal sequence

functions as an export signal that resembles the bacterial export

signal and targets the intermediates from the matrix across the IMM

into the IMS.

As already mentioned, the Qcr7 protein does not contain a

cleavable amino-terminal signal sequence and it is not known which

pathway it follows t o reach its final destination in the IMM.

However, it probably follows the general matrix pathway, since it

requires extra- and intramitochondrial ATP as well as the membrane

potential for import (1 42). Furthermore, there is circumstantial

evidence that the Qcr7 protein does not follow the import pathway

of proteins without a cleavable signal sequence such as the

ADP/ATP carrier (143). In addition, subunit 7 is believed to assemble

into a subcomplex with cytochrome b and the 1 1 kDa subunit (33. 60).

Although it is not certain at present whether this subcomplex

assembles directly in the membrane or first in the matrix before

insertion into the IMM, the latter hypothesis seems to be favored

according to the findings of a recent study. Japa e t al. (142) have

shown that the Qcr7 protein is not bound to the membrane when

performing in vitro import studies with mitochondria from a strain

lacking cytochrome b. When performing the same experiment with

mitochondria from wild type yeast, however, the Qcr7 protein was

bound to the IMM through protein-protein interactions. Hence, the

formation of a subcomplex containing cytochrome b, the 14 kDa and

the 11 kDa subunits probably occun in the matrix and a targeting

sequence must therefore be contained in the Qcr7 protein. However,

it cannot be ruled out completely that the Qcr7 protein is imported

into the IMM without passing through the matrix and, in the absence

of cytochrome b, passes on to the matrix.

The third possible function for the Qcr7 protein that I

investigated was the role of the subunit 7 N-terminus in the

assembly of ubiquinol-cytochrome c oxidoreductase. Previous

studies indicated that deletion of the QCR7 gene resulted in low

steady-state levels of the ISP, cytochrome b, and the 1 1 kDa protein

(33. 60). Due t o a post-translational interdependence of cytochrome

b, the 14 kDa subunit, and the 11 kDa subunit, which has been

demonstrated a number of times, it is believed that a subcomplex

consisting of these three components is formed prior to assembly of

these subunits into a functional complex 111 (33. 60-63). The ISP does

not seem to be a component of this subcomplex since a mutant strain

containing a deletion of the gene encoding the ISP does not contain

lowered steady-state levels of complex III subunits other than the

ISP itself (60).

To test the involvement of the Qcr7 protein N-terminus in

proton pumping, rnitochondrial targeting, and assembly of complex

111, 1 mutated the QCR7 gene in regions corresponding to the

following residues (refer to Fig. 3-2). A previous study by lmoto e t

al. suggested that a number of serine and threonine residues of the

nicotinic acetylcholine receptor probably form a ring or hydrophilic

pore structure that comes into close contact with permeating

cations and may determine the selectivity of the channel (165). In

addition, a study performed by Yool and Schwarz also implicated a

role for serine and threonine residues in the formation of a K+

channel (166). Similarly, in solving the crystal structure of the beef

heart mitochondrial cytochrome oxidase Tsukihara e t al. found that

one of the channels involved in H+ pumping terminated a t two serine

residues that may be involved in hydrogen bonding (168). Based on

these studies and the knowledge that mitochondrial signal sequences

are enriched with hydroxylated residues (939 941, such residues in the

N-terminus were targeted for mutagenesis to investigate their role

in import and in the possible construction of a H+ channel. Since,

however, mutating Ser-4 and Thr-6 did not affect mitochondrial

import or H+ translocation, and since a protein with an N-terminal

truncation of 20 residues is imported into the mitochondria to the

same extent as a protein with a truncation of 7 residues, no

additional hydroxylated residues were mutated (see Chapter 4).

Four mutants with Qcr7 proteins truncated by 7, 10, 14, and 20

residues from the mature protein (that lacks Met-1, due to cleavage)

N-terminus were also tested for their role in H+ translocation, in

accordance with the results from the studies in beef heart

mitochondria. Since the yeast 14 kDa subunit contains a longer N-

terminal extension than i t s beef heart homologue, deletion of 20

residues was comparable to the proteolytic cleavage (7 to 11

residues) that occurred from the N-terminus of the beef heart

subunit (131, 141) (Fig. 3-2). Contrary to the studies performed in

beef heart mitochondria, the introduction of deletions into the

plasmid-borne QCR7 gene enabled me to study the involvement of the

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* A * * * * * * + * * X -ic

Y: E S Y A R A Y R I I R A H Q T E L T H A L L P R N Q W I K F F Q E B : L Y D D R V F R I K R A L D L s M R Q Q I L P K E Q W T K Y E E

Fig. 3-2. Alignment of yeast Qcr7 protein and its 13.4 kDa homologue from beef heart mitochondrial cornplex III; adapted from Link e t al. (23). Y = yeast Qcr7 protein ( 1 4.5 kDa sobunit 7); 8 = beef heart 13.4 kDa subunit. * = conserved residues; + and - indicate conserved charges, and A indicates an exchange of one aromatic residue for another. Truncations of 7, 10, 14, and 20 residues from the N-terminus of the mature Qcr7 protein (which lacks Met-1) are indicated by arrows. Individual residues that were substituted as a result of mutagenesis are underlined.

Qcr7 protein N-terminus (in events prior t o and involving the

assembly of the complex) in vivo rather than in vitro.

The Qcr7p-~7 displays a phenotype that is comparable t o the

wild type a t 30°C. Hence, a number of point mutations were

introduced into the QCR7 gene in the context of a a7 deletion.

Arginine-10 was targeted for two reasons. The first one, is t o test

i ts involvement in mitochondrial targeting since arginines are

typical for mitochondrial signal sequences. The other reason for

mutating Arg-1 O, which was also the reason for mutating Asp-13,

was to find out whether these amino acids might be important for

assernbly by providing contact with another subunit through salt-

bridge formation. Full-length mutants containing Qcr7 proteins with

substitutions for Arg-1 O and Asp-13 were created to investigate

the possible role of these residues in import and assembly of the

Qcr7 protein.

Of the other substitutions shown in Fig. 3-2, substitutions in

Gln-31 were created t o test the earlier hypothesis that the Qcr7

protein is involved in ubiquinone-binding. Although this hypothesis

was later corrected in the literature, as it was based on a mistake,

mutants with Qcr7 proteins containing residue substitutions for

Gln-31 had already been tested for complex Ill-linked activities and

ATP synthesis and found to be normal. Hence. Gln-31 is most likely

not involved in ubiquinone-binding. Full-length mutants with Qcr7p-

N53S and Qcr7p-N53D were created t o investigate the pet-

phenotype of the mutant with the truncated protein Qcr7p-

~7(N53S/E116G). Residue Glu-1 1 6 was not assessed as a cause for

the pe t - phenotype as a previous study, in which a segment

containing this residue was replaced by three amino acids encoding

STOP codons in al1 reading frames, did not result in a respiration-

deficient mutant (49). However, the levels of apo-cytochrorne b, the

ISP, and the 11 kDa subunit are reduced in this mutant. In addition,

complex Ill-linked enzyme activities are reduced by approximately

40%, whereby the mutant enzyme retains a normal turnover number.

These findings of the study by Hemrika e t al. (49) implicate the Qcr7

protein C-terminus in assembly. All the other residue substitutions

arose fortuitously during mutagenesis and were also investigated

for causing phenotypes relevant to this study.

The current chapter describes the mutagenesis of the QCR7

gene followed by a prelirninary characterization of the mutant

strains according to growth and enzyme activities. In total, 22

different residues were substituted and they were changed to 38

different amino acids. In addition, four truncated proteins were

constructed which were missing the N-terminal 7, 10, 14, and 20

residues, respectively. The protein with the N-terminal truncation

of 7 residues was further subjected to point substitutions, whereby

a total of six such proteins were constructed, four of which were

multiple mutants. In total, 38 individual mutant proteins were

constructed and the strains expressing these were further analyzed.

A detailed analysis of the role of the Qcr7 protein N-terminus with

respect to proton pumping, mitochondrial import, and assembly of

complex III is given in Chapter 4.

3.2. MATERIALS AND METHODS

Ma terials

Oligodeoxynucleotides were synthesized by the Department of

Clinical Biochemistry a t the University of Toronto. Restriction

enzymes were purchased from Pharmacia. The T7 GEN in vitro

mutagenesis k i t was obtained from United States Biochemical

(Cleveland, OH). The pG-3 expression vector ( i l * ) was a kind gift

from Dr. Jacqueline Segall a t the University of Toronto. This vector

contains a 2p origin for replication in yeast as well as pUC18

sequences for replication in bacteria and ampicillin resistance.

Selection in yeast occurs by the TRP-1 gene marker. Expression of

the insert is driven by the glyceraldehyde-3-phosphate

dehydrogenase promoter and termination as well as polyadenylation

signals are from the phosphoglycerokinase gene.

Mutagenesis by PCR

Oligodeoxynucleotides were synthesized containing mutations

in codons for the N-terminal portion of the Qcr7 protein (Table 3-1)

and a Kpnl restriction site a t the 5' end. Together with a wild type

oligodeoxynucleotide downstream of the QCR 7 coding reg ion t o

which a Sall restriction site was added, mutant genes were

amplified using PCR as described in Chapter 2. The original pG-3

expression plasmid was modified by digestion with Sacl t o excise a

1700 bp fragment in the polylinker region and subsequently self-

ligated to create the new p G - 3 ~ vector. PCR products were digested

with Safi and Kpnl and then subcloned into the respective sites of

Table 3-1. Oligodeoxynucleotide sequences used for mutagenesis of the QCR7 gene. OIigodeoxynucleotides were designed with deletions or base substitutions in the QCR7 gene. Following PCR amplification or M l 3 mutagenesis, the wild type codon sequence was substituted by a sequence containing a deletion or a missense mutation. Abbreviations used are: n=a, c, g, t; z=a, c, t; x=a, c, g, and "/" stands for "or1'.

Table 3-1. Primen Used for Mutagenesis

I Primer Sequence (5'03')

ccggtaccatggcgazaattgg tgacP

gtcaccaattg/atcgcaatagacgu

ccggtaccatggcgagaattggtga/tgtatattttgaagtcacccP

ggtgacttcaaaatatactc/taccaattctcgM

tctgcatgatgggagattcctctgcaM Oligonucleotides used for mutagenesis by PCR. Oligonucieotides used for Ml 3 mutagenesis.

Mutat ion/s

Encoded

wild type for 3'end

A 7

A1 O

A1 4

A20

R101, K, T (67)

RI 01, T

D l 3E, V ( ~ 7 )

D13E, K

S4A, C, Dl G, H, L, Y

T6P, Q R, S

Q31L, S, WQ

N34SQ

N53iY

N53Sç

These mutations were introduced to test an earlier hypothesis according to which the Qcr7 protein was believed to be a ubiquinone-binding protein. However, this was a mistake in the literature which has since been corrected.

This residue was mutated to investigate the pet- mutant expressing Qcr7p ~7(N53S/E116G).

the pG-3a vector. lnserts were sequenced as described in Chapter 2.

Yeast strain YSM-qcr7~ was transformed as described by Hill e t al.

(137) with pG-3A plasmids containing mutated qcr7 genes.

Site-directed mutagenesis using the Ml 3 phage

Oligodeoxynucleotides were synthesized containing various

nucleotide changes (Table 3-1). M l 3 mutagenesis was performed

using the T7 GEN in vitro mutagenesis kit and al1 manipulations were

performed as suggested by the manufacturer. For this technique the

QCR7 gene was subcloned into the double stranded, replicative form

RFM13, of the Ml 3 phage. The QCR7 insert was subsequently used in

the single stranded form of the M l 3 phage as a template for

mutagenesis. In the in vitro mutagenesis, primers were annealed to

the template and extended by T7 DNA polymerase to synthesize a

second strand of the QCRP gene-containing M l 3 phage. During the

second strand synthesis, 5'-methyl dCTP was incorporated into the

new strand. This ensured that the newly synthesized, methylated

strand was preserved whereas the original strand was nicked by

treatment with Mspl and M a l and subsequently degraded by

exonuclease III. Following mutagenesis, single stranded phage DNA

was prepared and sequenced t o identify mutated qcr7 genes. Mutated

genes were subsequently isolated from the RFM13 phage (139) and

transferred to the modified yeast expression vector pG-3A. The pG-

3 ~ - g c r 7 plasmids were transformed into strain YSM-qcr7A (1 37).

Enzyme assays

Mitochondria were isolated frorn glucose-grown cells. NADH-

cytochrome c reductase (complexes I+III) and succinate-cytochrome

c reductase (complexes II+III) activities were assayed in 0.1 M

potassium phosphate buffer (pH 7.0)-94 pM cytochrome e l mM

sodium azide containing between 20 and 100 pg of mitochondria. To

start the reaction, 34.2 pM NADH or 10 mM sodiumsuccinate were

added as substrates, respectively, and the reduction of cytochrome c

was monitored spectrophotometrically a t 5 50 nm. Cytochrome

oxidase activities were assayed in 0.1 M KPi buffer (pH 7.0) with

reduced cytochrome c as substrate (94 PM). Reduced cytochrome c

was prepared by the addition of 3.2 mM ascorbate to a suspension of

oxidized cytochrome c (40 mg/mL) followed by dialysis against 0.1

M KPi for 48 h during which the buffer was changed twice.

Mitochondria (20-50 pg) were added to start the reaction and the

oxidation of cytochrome c was followed a t 550 nm. A number of

ubiquinol-cytochrome c reductase assays (complex III) were

performed to show that the observed succinate-cytochrome c

reductase and NADH-cytochrome c reductase activities were a true

reflection of the technically more difficult complex III assays. For

these assays decylubiquinol, which was prepared f rom

decylubiquinone by reduction with HCI, was used as the substrate in

0.1 M KPi-94 p M cytochrome c-1 mM azide (144).

Gro wth s tudies

Growth was tested on solid medium a t RT (20-230C), 30oC, and

370C. To identify respiration deficient mutants, cells were grown

on SD medium containing ethanol (4%) and glycerol (3%) as the main

carbon sources with the addition of a small amount of glucose

(0.1 46). Mutant strains that were p e t at 300C were picked and rnated

with a rho- strain for further selection by growth on SD medium

containing only the non-fermentable carbon sources glycerol and

ethanol. Respiration deficient mutants that contained mutations in

the mitochondrial genome failed t o grow under these conditions and

could thus be eliminated from the selection.

3.3. RESULTS

Characterization of mutants by gro wth

Mutant versions of the Qcr7 protein were assessed for their

ability t o support respiration-dependent growth. This was

performed by site-directed mutagenesis of the QCR7 gene using

either the PCR or M 1 3 mutagenesis technique (see Materials and

Methods) and the oligodeoxynucleotides listed in Table 3-1 . Plasmid-borne versions of the QCR7 gene were introduced into the

strain containing the chromosomal qcr7A:LEUZ allele, YSM-qcr7A,

and characterized according to their growth patterns at RT (20-

23oC), 30C, and 37oC on two types of solid media. Synthetic

deficient medium containing ethanol and glycerol as main carbon

sources with the addition of 0.1% glucose is typically used to

identify respiration deficient strains in yeast. Hence, respiration

deficient strains arising from mutated Qcr7 proteins that affect

complex III function grow on this medium until the small amount of

glucose is depleted, but only small colonies form which are referred

to as pet- mutants. Strains containing mutations in the qcr7 gene

which do not affect function, on the other hand, will grow to wild

type size colonies. Such strains can continue to grow by

catabolizing ethanol and glycerol once glucose has been depleted.

Similarly, respiration deficient strains remain white when grown on

SD medium containing glucose as the sole carbon source, while cells

with a functional respiratory chain assume a red phenotype. This

red phenotype is due to the ade2 mutation that is present in the

parental strain W303-1B and causes a red pigment to accumulate

(14s). In respiration deficient strains, however, this pigment does

not accumulate and the cells retain a white phenotype. Hence, p e t

strains can be clearly distinguished from wild type strains on the

basis o f their growth characteristics on these types o f media. The

growth characteristics of selected strains are shown in Fig. 3-3 and

the results of the analyses of al1 38 mutant strains are summarized

in Table 3-2. The strain expressing Qcr7p-67 had wild type growth

characteristics at RT and 30oC, but was p e t - a t 370C. This

conclusion was based on the observations that a t RT and 300C this

strain formed wild type shed colonies on SD medium containing

ethanol and glycerol as the main carbon sources (top panel) and the

colonies were red on SD medium containing glucose as the sole

carbon source (bottom panel). In contrast, the strains expressing

Qcr7p-~10, Qcr7p-dl4 and Qcr7p-A20 were respiration deficient at

al1 temperatures. These strains formed small colonies on the non-

fermentable carbon sources (Fig. 3-3, top) and the colonies were

white on glucose (Fig. 3-3, bottom). Strains containing Qcr7p-

~ 7 ( R 1 0 K ) and Qcr7p-~7(A9V/R1 OT/Y14N/N53D) behaved as did

Qcr7p-a7; that is, they were pet - at 370C but not a t RT or 300C. In

contrast, Qcr7p-~7(RI OT/K44N), Qcr7p-~7(RI OI/G1 ZV), Qcr7p-

~7(D13V) , and Qcr7p-~7(N53S/E116G) were pet- at al1 temperatures

tested. A t 370C, al1 mutants including proteins with an amino-

terminal deletion, even those which displayed wild type

characteristics a t RT and 300C, were respiration deficient. None of

the full-length genes containing point mutations were found t o

cause deficiencies at any of the three temperatures.

Fig. 3-3. Growth on SD agar plate with ethanol/glycerol and 0.1% glucose, or SD media plate containing glucose. Top: when grown primarily on the non-fermentable carbon sources ethanol and glycerol, the mutant strain containing the Qcr7p-~7 is comparable in size to the wild type. Mutant strains expressing Qcr7 proteins truncated by 10, 14, or 20 amino acids are pet- mutants, indicative of a respiratory chain defect. Bottom: when grown on SD medium containing glucose as the sole carbon source, respiration competent strains turn red due to the ade 2 mutation which causes a red pigment t o accumulate. Strains expressing Qcr7 proteins truncated by 10, 14, and 20 residues remain white, confirming severe respiration defects.

Enzyme activities

I next tested whether the strains expressing mutated qcr7

genes had functionai deficiencies o f ubiquinol-cytochrome c

oxidoreductase. For this experiment, I had planned to grow cells on

a carbon source such as galactose, raffinose, or maltose, since these

sugars do not lead to catabolite repression. I discovered, however,

that pet- mutants did not grow well on minimal medium containing

these sugars, presumably because they were too compromised.

Hence, I performed enzyme assays with mitochondria from cells

grown in glucose. This was less than ideal, since glucose resulted in

significant catabolite repression of the QCR7 gene and most likely

the genes encoding the other subunits of the bci complex. The

activity of complex III was drastically lower in glucose grown cells

relative to cells grown in galactose. Nonetheless, it was possible to

compare complex Ill-linked activities in mitochondria from glucose-

grown cells.

Complex III-lin ked enzyme activities were measured with

mitochondria purified from each of the mutants grown a t 300C.

Complex III was either measured in combination with complex 1,

using NADH as substrate, or in combination with complex i l , using

succinate as substrate. The results obtained from the enzyme

assays correlated with the results from the growth studies, i.e., al1

mutants that were characterized as pe t - mutants in the growth

studies, displayed a severe decrease in complex Ill-linked enzyme

activities. Cytochrome c oxidase activities were measured as a

control t o determine the integrity o f the mitochondria. I found that

in al1 pet- mutants cytochrome c oxidase activities were decreased

by about 40% when compared to the wild type. This decrease in

complex IV activity can be explained by a downregulation of the

expression of mitochondrial respiratory chain proteins due to a

respiratory chain defect. Results are summarized in Table 3-2.

Table 3-2. Summary. Characterization of mutants according to growth and enzyme activities. Growth was monitored a t three temperatures: RT, 30°C, and 37OC. Mutant strains carrying plasmid-borne versions of qc r7 are named according t o their mutations in the Qcr7 protein and are classified with regard t o whether they cause a pet- phenotype (pet) or not (+). Complex III- Iinked enzyme activities were measured and the mutants were divided into two classes. One class of mutants had wild type level enzyme activity (wt), and the activity of the other class was as low as in YSM-qcr7~ ( 0 ) . Complex IV enzyme activities from mutants were divided into two classes. One class had wild type activity (wt) and the other class had activity of about 60% that of wild type. Typical enzyme rates are: complex Ill-linked wild type, 30-35 +/-5 nmol min-' mg"; complex Ill-linked activities for YSM-qcr7A, O nmol min-' mg-'; complex IV activity of wild type, 125 +/- 11 nmol min-' mg-'.

MUTANT

A 7 R1 OK (~7) R1 OI/G72V (~7) R1 01 R I OT/K44N (~7) A9V/R1 OT/ Y74N/N53D (~7) R I OT D l 3V (~7) D l 3K D l 3E N53S/E 1 7 6G (~7) N53S N53D 11 0 $1 4 120 s4c 340 54G 34H 34Y 54WP2T 54G/E 7 O9G 54NL35P ;4y/17 7 v ;4G/P2Q r6P r6Q -6 R -6s 231 L 13 1 s 13 1 W C 7 7N WS/I 7 07V 4 9 v ! 78s 199N IO plasmid (strain YSM-qcr7a)

Italicized mutations arose for chain defect.

- RT

- + +

Pet +

Pet + +

Pet + +

Pet + +

Pet Pet Pet + + + + + + + + + + + + + + + + + + + + + + pet

30OC

- + +

Pet +

pet + +

Pet + +

Pet + +

Pet pet Pet + + + + + + + + + + + + + + + + + + + + + + pet

Complex III linked

act ivity wt wt

trace wt -

wt wt -

wt wt - wt wt - - -

wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt -

Complex IV activity

tousiyand were also tested for causing a respiratory

3.4. DISCUSSION

This chapter describes the introduction of a number of point

and deletion mutations into various regions of the QCR7 gene with an

emphasis on the portion corresponding t o the Qcr7 protein amino-

terminus. These mutant genes, contained in an expression vector,

were subsequently expressed in yeast and the phenotype of the

resulting strains was assessed. Classification o f al1 mutants

according to growth and enzyme activities divided them into three

categories: mutants with wild type characteristics, mutants that

resemble strain YSM-qcr7~, a n d mutants that had wild type

characteristics a t RT and 30oC and characteristics like YSM-qcr7~ a t

37°C (Table 3-2). Notably, neither the strain expressing Qcr7p-~7,

nor any of the strains containing full-length Qcr7 proteins with

point mutations were deficient at 3OoC. However, strains

expressing deletion proteins Qcr7p-Al O, Qcr7pA14, and Qcr7p-a20

as well as Qcr7p-A 7(Rl OT/K44N), Qcr7p-b7(R1 OI/G12V), Qcr7p-

~7(D13V), and Qcr7p-b7(N 5 3 S/E1 1 6G) were respiration-deficient a t

al1 temperatures tested.

Many mutants displayed the inability t o grow in media

containing non-repressive carbon sources such as galactose,

raffinose, or maltose. When yeast are grown on high levels of

glucose, many respiratory chain related proteins suffer catabolite

repression; this means that the uptake systems of sugars other than

glucose are repressed (Z 15). ln order to compare results between

wild type and mutants, al1 strains had t o be grown in the same

medium, hence this medium had to contain glucose as the carbon

source. Unfortunately, I found that growing the cells on glucose

resulted in a significant catabolite repression of the QCR7 gene.

This was evidenced by a lower level of the QCR7 RNA transcript in

glucose-grown cells as opposed to galactose-grown cells (results

not shown). Catabolite repression of the QCR7 gene also led t o

lowered complex Ill-linked enzyme activities; this might indicate

that al1 or some of the genes encoding the other subunits of the bci

complex are also repressed.

As can be seen from the results summarized in this chapter,

despite 50 mutations that were introduced into the QCR7 gene only

very few resulted in a respiration defect. In addition, none of the

full-length Qcr7 proteins containing point mutations resulted in a

deficiency. It might therefore be more instructive to target highly

conserved residues in the future, especially with the use of a recent

cornparison of homologues to the Qcr7 protein from a wide variety of

organisms (146). Alternatively, it might be more successful to "work

backwards"; for example, one could select for respiration-deficient

mutant strains on non-fermentable carbon sources following random

mutagenesis of the QCR7 gene. A random mutagenesis approach was

not used for this study as I was particularly interested in

determining the function of the Qcr7 protein amino-terminus with

respect to proton translocation, mitochondrial import, and assembly

of complex III (see Introduction of this Chapter). In addition,

truncation of seven residues from the N-terminus of the Qcr7

protein resulted in the same phenotype, but a different profile of

subunits from the wild type and indicated that the N-terminus is

essential. This prornpted me to concentrate on the amino-terminus.

lmmunoblotting established that deleting as many as 20

residues from the Qcr7 protein N-terminus did not prevent this

subunit from being imported into mitochondria (results shown in

Chapter 4). Hence, the preliminary analysis of the respiration-

deficient mutants described in this chapter indicates that the

amino-terminus of the Qcr7 protein is essential for the functioning

of ubiquinol-cytochrome c oxidoreductase. A detailed

characterization of the role of the Qcr7 protein amino-terminus in

assembly of complex III, proton pumping, and mitochondrial import

is given in Chapter 4.

CHAPTER 4

The Role of the Amino-terminus

of the Qcr7 Protein in

Mitochondrial Targeting,

Complex I I I Assembly,

and

Proton Pumping

Contribution to this chapter:

Joses Jones perforrned the immunoblotting of figure 4-7.

CHAPTER 4

The Role of the Amino-terminus of the Qcr7 Protein in

Mitochondrial Targeting, Complex III Assembly, and Proton

Pumping

4.1 . INTRODUCTION

The mutational analysis described in Chapter 3 indicated that

the amino-terminus of the Qcr7 protein is essential for formation of

a functional complex III. Hence, with some pet - mutants now

identified I proceeded to test whether the amino-terminus of the

Qcr7 protein is involved in mitochondrial targeting and/or assembly

of ubiquinolsytochrome c oxidoreductase.

The involvement of the Qcr7 protein amino-terminus was also

investigated with respect to proton translocation. This hypothesis

was tested by indirectly measuring the integrity o f the proton

gradient which is only established under conditions where electron

transport is functional. Hence, only strains which contained a fully

or partially functional enzyme cornplex were tested; these included

strains expressing Qcr7 proteins with mutations in Pro-2, Ser-4,

Thr-6, and Ile-11, as well as the strain expressing Qcr7pA7.

4.2. MATERIALS AND METHODS

Ma terials

Chemicals were purchased from Sigma (St. Louis, MO), BDH

(Toronto, ON), or BioRad (Hercules, CA). Dodecylmaltoside was

purchased from Boehringer Mannheim (Mannheim, Germany), and

dithiobis(succinimidyl propionate) (DSP) from Pierce (Rockford, CT).

Restriction enzymes and protein A sepharose were obtained from

Pharmacia (Montreal, PQ). Anti-Qcr7 protein antibodies used for

immunoprecipitation were raised in chickens and rabbits against a

carboxy-terminal peptide of the protein (refer t o Chapter 2).

Antibodies raised against subunits of the bel complex were a kind

gift from Dr. Bernard L. Trumpower a t Dartmouth Medical School. An

antibody t o yeast S-acetyl coenzyme A synthetase was raised in

rabbit by the Centers of Excellence (Canada). Goat anti-mouse IgG

(H+L) coupled to alkaline phosphatase and goat anti-rabbit IgG (H+L)

coupled t o alkaline phosphatase were purchased from BioRad.

Alkaline phosphatase-conjugated AffiniPure rabbit anti-chicken IgY

(IgG) (H+L) was purchased from Jackson lmmuno Research

Laboratories, Inc. (Westgrove, CT). BioMag magnetic beads were

obtained f rom Perseptive Diagnostics (Cambridge, MA).

Isolation of yeast mitochondria and Western analyses

Mitochondrial isolations and Western analyses were

performed as outlined in Chapter 2.

A TP synthesis assays

Freshly prepared mitochondria (approximately 50 pg) were

resuspended in 200 PL breaking buffer (0.6 M sucrose, 20 mM HEPES-

KOH, pH 6.5, 0.1% BSA) without PMSF. To start the reaction, the

mitochondria were added to 5 mM KPi (pH 7.41-1 mM ADP-5 mM

succinate. The mixture was incubated at 370C for 45 min and the

reaction was stopped with 80 mM perchloric acid. Proteins were

pelleted and the supernatant was assayed for the amount of ATP

synthesized by using a hexokinase/gIucose-6-phosphate

dehydrogenase coupled assay (147) in which NADPH is generated.

NADPH was quantitated in an Eppendorf fluorimeter.

C W a r Dichroism spectra

Peptides were chosen from the N-terminus of the Qcr7 protein

in yeast and its homolog in beef heart and synthesized by the Alberta

Peptide Institute:

AGRPAVSASSRWLEG (residues 2-1 6, beef heart subunit 6;

peptide 1 );

AGRPAVSASSRWLEGIRKWYYNAAG (residues 2-26, beef heart

subunit 6; peptide 2);

PQSFTSIARIGDY (residues 2-1 4, Qcr7 protein; peptide 3);

PQSFTSIARIGDYILKSPVLSKL (residues 2-24, Qcr7 protein;

peptide 4).

For recording CD spectra, peptides were dissolved at 1 mg/mL in

either 10 mM NaCI-10 mM NaH2P04, methanol, or SDS (30-fold molar

excess) diluted in the above buffer. Spectra were recorded on a

Jasco J-720A spectropolarimeter, each with two to three scans

from 250 to 190 nrn a t 250C. Baseline spectra for each solvent were

subtracted from the peptide spectra.

Spectra of the cytochromes

For spectral analyses of the cytochromes, rnitochondria were

resuspended in 0.1 M potassium phosphate (pH 7.4)-0.25 M sucrose-

0.5% cholic acid (33). TO obtain a spectrum containing cytochromes c

and c l , cytochrome b, and cytochromes a and a,, a ferricyanide-

oxidized spectrum was subtracted from a dithionite-reduced

spectrum. To obtain a spectrum containing cytochrome b only,

dithionite-reduced minus ascorbate-TMPD (0.2 mM) reduced samples

were run. Spectra were recorded on a DW-2a Aminco

spectrophotometer from 520 to 620 nm.

Co-immunoprecipita tion

Different immunoprecipitation protocols were performed with

various combinations of the buffers and procedures listed below.

When the anti-Qcr7 antibody raised in chicken was used as the

primary antibody, magnetic beads had to be employed since Protein A

Sepharose does not bind chicken lgG. On the other hand, when

imrnunoprecipitations were performed with the anti-Qcr7 antibody

raised in rabbit, protein A sepharose was employed as the matrix to

bind the primary antibody. lmmunoprecipitated proteins were

analyzed by immunoblotting under both reducing and non-reducing

conditions.

Buffen: a) NET-gel buffer (50 mM Tris-CI pH 7.5, 150 mM NaCI, 0.1 %

Nonidet P-40, 0.25% gelatin, 1 mM EDTA pH 8.0, 0.02% sodium

azide), containing 0.05% to 1% Nonidet P-40 (139)

b) 50 mM Tris-HCI (pH 8), 1 mM MgS04, 0.5 mM PMSF, 0.8 g

dodecylmaltoside/g protein

c) 250 mM sucrose, 1 mM EDTA (pH 8), 10 mM MOPS-KOH (pH

7.2) , 60 mM KCI, 0.5 mM PMSF, 0.7% digitonin

d) 50 mM Tris-HCI (pH 8), 1 50 mM NaCI, 1 mM EDTA (pH 8),

0.25% gelatin, 0.5 mM PMSF, 0.8 g dodecylmaltoside/g

protein

e) 5 0 mM Tris-HCI (pH 8), 1 50 mM NaCI, 5 mM EDTA, 0.5

mM PMSF, 0.8 g dodecylmaltoside/g protein

Procedures;

A) Preclearing: mitochondria (100-200 pg) solubilized in one

of the above buffers, were incubated with a) pre-immune serum, or

b) rabbit-anti-yeast S-acetyl coenzyme A synthetase a t 0°C for

approxirnately 60 min. Subsequently, protein A sepharose or

magnetic beads were added and the suspension was incubated for

another 30 min a t O°C. Beads were pelleted, and the supernatant

fraction was used further for imrnunoprecipitation.

Immunoprecipitation: mitochondria-containing supernatant

(from above) was rotated a t 0°C with varying concentrations of

anti-Qcr7 protein antibody. Subsequently, protein A sepharose or

magnetic beads (coupled to rabbit-anti-chicken antibody), were

added and the suspension was rotated at 4°C for 1 h. The beads were

pelleted and washed three times with buffer by rotating for 20 min

during each wash. After the final wash, al1 o f the liquid was

removed from the beads which were subsequently resuspended in 30

pL of SDS-PAGE buffer with or without DlT, heated for 3 min a t

95°C and analyzed by immunoblotting.

B) Mitochondria solubilized in one of the above buffers (5

rng/mL) were incubated on ice for 10 min, centrifuged a t 4*C for 10

min in a microcentrifuge to remove insoluble materials, and

transferred to Protein A Sepharose beads/BioMag magnetic beads.

Subsequently, the volume was increased t o 1 mL and the

mitochondria-bead suspension was rotated for 1 h a t 4°C to reduce

the concentration of proteins in the suspension which bind the

matrix in a nonspecific manner. The beads were pelleted and the

supernatant was transferred to a new bead suspension, after which

the primary antibody was added and the suspension was incubated

overnight with rotation a t 4°C. Samples were washed three times in

buffer, beads were resuspended in 2 x SDS-PAGE buffer not

containing DTT and analyzed by immunoblotting.

Cross-linkina: -

Mitochondria (200 pg) were resuspended in 450 pL breaking

buffer (pH 8.0) containing 0.5 mM PMSF and 10 mM iodoacetamide.

The cross-linking agent DSP was added from a 500 mM stock in dry

DMSO to a final concentration of 0.28 mM, the suspension was

incubated on ice for 20 to 30 min, and the reaction was terminated

with 5 0 pL of 100 mM glycine containing 10% aprotinin.

Mitochondria were pelleted and washed once with breaking buffer

containing 0.5 mM PMSF. lmmunoprecipitations using procedure (B)

with various buffers from the list above followed the cross-linking

procedure devised by Dr. David B. Williams at the University of

Toronto. lmrnunoprecipitated proteins were analyzed by

immunoblotting (as described in Chapter 2) under reducing and non-

reducing conditions.

4.3. RESULTS

Circular Dichroism spectra of amino-terminal peptides

Link e t al. (23) have previously suggested that the N-terminus

of the 13.4 kDa subunit 6 of complex III from beef heart

mitochondria forms an amphipathic a-helix. To confirm this

helicity, and t o compare the yeast N-terminus of the Qcr? protein to

i ts homologue in beef heart mitochondria, two peptides

corresponding to amino acids 2-16 and 2-26, respectively, for the

bovine sequence and 2-1 4 and 2-24, respectively, for the yeast

sequence were synthesized (see Methods section).

CD spectra were obtained for al1 four peptides in diluted saline

buffer, methanol, and SDS micelle suspension in saline buffer (Fig.

4-1). All peptides formed only a Iimited amount of secondary

structure in aqueous buffer. However, as shown in Fig. 4-1 (middle

panels, top and bottom) by the minima at 208 nrn and 222 nrn which

are characteristic for a-helix formation, peptides 2 (long beef heart

peptide) and 4 (long yeast peptide) display considerable a- he l i x

content in methanol. In SDS micelles, the secondary structure of

peptides 2 and 4 are similarly a-helical. Spectra and helical

content of peptide 4 strongly resemble that of peptide 1 (shon beef

heart peptide, spectra not shown). This indicates that although the

N-terminal regions of the yeast and beef heart proteins are not very

similar (due to the increased length of the yeast N-terminus), they

nevertheless display similar secondary structures. Peptide 3 (short

yeast peptide), on the other hand, did not display any a-helix

Fig. 4-1. Circular Dichroism spectra. Peptides corresponding t o amino acids 2-26 of the beef heart 13.4 kDa subunit (peptide 2) and amino acids 2-24 of the yeast 14.5 kDa subunit (peptide 4) were assayed for secondary structure by CD. Peptides were dissolved a t a concentration of 1 mg/mL in buffer (1 0 mM NaCI-10 mM NaH2P04), methanol, or SDS (to a 30-fold molar excess diluted in above buffer). Spectra were recorded a t 250C on a Jasco J - 7 2 0 A spectropolarimeter. The y-axis represents the mean residue molar ellipticity which contains the units [deg cm2 dmoïl]. Top three panels, beef heart peptide 2; bottom three panels, yeast peptide 4.

2 4 ' A 4

iu ô g g g b b 0 0 0 0 0 0 ~

T ' T Y Y ' + + T ' O O O O O O O O N N N N O W

formation in water, methanol, or SDS (not shown). This indicates

that although many peptides display helical spectra in SDS

independent of their role in vivo, one can a t Ieast conclude from

these experiments that the peptides involved possess the intrinsic

capability ("threshold hydrophobicity" coupled with residue-

dependent helical propensity) to penetrate membranes and form

stable secondary structures (1 48).

The amino-terminus o f the Qu7 protein may facilita te import into

mitochondria

Many nuclear encoded mitochondrial proteins possess targeting

sequences, 15-70 amino acids long, that are usually located a t the

N-terminus. Some proteins such as cytochromes c, and b2 even have

bipartite signal sequences that are cleaved in two steps (12& 149).

Mitochondrial targeting sequences have no obvious homology, but are

generally rich in hydrophobic and hydroxyla ted a mino acids, have a

net positive charge, and are able to forrn amphipathic a-helices

when in contact with the lipid bilayer (6. 50. ' s i 1.

The Qu7 protein does not contain a cleavable N-terminal

mitochondrial targeting sequence (21, but according to Sirrenberg e t

al., it is also unlikely to follow the import pathway used by proteins

without a cleavable amino-terminal signal sequence such as the

ADP/ATP carrier (143). Since the Qcr7 protein N-terminus contains

many residues that are typical of mitochondrial signal sequences,

and since CD spectrophotornetry of an N-terminal peptide has shown

that there is a potential for forming an a -helix (Fig. 4-1) with

Fig. 4-2. Helical wheel projections. The amino-terminal 18 residues (starting after Met-1) are plotted. a) Wild type; b) Qcr7 protein truncated by residues 2-8 from the N-terminus. A helical wheel projection of the wild type shows that al1 the charged and most of the polar residues are located on one face of the helix, whereas the majority of the hydrophobic residues are located on the opposing face. A helical wheel projection of the Qcr7p-a7 shows that charged residues are located on one face of the helix whereas hydrophobic and polar residues are interspersed throughout.

amphipathic character for the wild type (Fig. 4-2), 1 decided to

investigate whether this region contains any information for the

mitochondrial localization of the protein. Accordingly, I tested the

mutants expressing Qcr7p-A7, Qcr7p-Al O, Qcr7p~14, and Qcr7pa20

(see Chapter 3, Fig. 3-2) for the presence of their respective

truncated Qcr7 proteins in the mitochondria. Mitochondria were

purified from YSM-qcr7A strains grown a t 30°C, each of which

expressed one of the above truncated proteins. Western blot analyses

of these mitochondrial proteins revealed that Qcr7 proteins lacking

7, 14, and 20 N-terminal amino acids are synthesized and

transporteci into mitochondria (Fig. 4-3, lanes 1, 2, and 4). However,

it was consistently found that the levels of truncated Qcr7 proteins

were reduced by approximately 60% when compared to the Qcr7

protein from the strain overexpressing the wild type gene (Figs. 4-3

and 4-4). These lower steady-state levels of the Qcr7 proteins

truncated by 7, 14, and 20 residues might be the result of a-helix

formation (if indeed they form a-helices) with compromised

amphipathicity as shown for Qcr7p-A7 (Fig. 4-2). Amphipathic

helices contain charged and polar residues on one face of the helix

and hydrophobic residues on the other. While this rule holds true for

the first 18 residues (after Met-1) of the wild type N-terminus, the

amphipathicity of Qcr7p-~7 (the N-terminus corresponds to residues

9 t o 26 of the wild type sequence) is compromised with hydrophobic

and charged residues interspersed throughout the helix (Fig. 4-2). In

addition, deletion of seven or more residues from the Qcr7 protein

N-terminus (after Met-1) results in the loss of the three

hydroxylated residues Ser-4, Thr-6, and Ser-7. Decreasing the

320 ~7 KO WT Core 1

- 0 .- - - - Core 2

Fig. 4-3. Western blot analyses of mitochondrial proteins from YSM-qcr7~ s t r a i n s overexpressing wild type and N- terminally truncated proteins Qcr7p-~7, Q c r 7 p - ~ l O, Qcr7p 61 4, Qcr7p-820. Mitochondrial proteins (100pg) were dissolved in SDS-PAGE buffer containing D l T and heated for 3 min a t 950C. Samples were run on 16% polyacrylamide gels and then transferred to nitrocellulose membranes. The blots on the top and middle panels were probed with an antibody recognizing core proteins 1 and 2, intermediate (i-ISP) and mature ISP (ISP), the 11 kDa and 14 kDa subunits. The blot containing cytochrome cl, on the bottom panel, was probed with a monoclonal antibody raised against this subunit. Top panel: Lane 1 : protein from YSM-qcr7~ overexpressing Qcr7p- ~ 2 0 ; Lane 2: protein from YSM-qcr7A overexpressing Qcr7pd7; Lane 3: protein from YSM-qcr7A; Lane 4: protein from YSM-qcr7A overexpressing wild type Qcr7 protein. Middle and bottom panels: Lane 1 : protein from YSM-qcr7A overexpressing Qcr7p-A 14; lane 2: protein from YSM-qcr7~ overexpressing Qcr7p-A20; lane 3: protein from YSM-qcr7~ overexpressing Qcr7p-Al O; lane 4: protein from YSM-qcr7a overexpressing Qcr7p-~7; lane 5: protein from YSM- qcrïA; lane 6: protein from YSM-qcr7~ overexpressing wild type Qcr7 protein.

% WILD TYPE 9) % WILD TYPE -

w % WILD TYPE w % WILD TYPE

Panel II. Number of blots used for histogram determinations

Subunit

14 kDa 11 kDa al1 ISP m-ISP a11 cyt cl m- cyt cl core 1 core 2 - - - 1 - -

* Due to faint bands on some of these blots, accurate quantifications could not be performed for these samples by densitometry. The numbers indicated reflect the number of blots inspected visually. The levels of 11 kDa subunit in the various mutant strains are as follows: WT, ~ 7 , R I OK > A I 4, 420 > D l 3V > R l OVG12V > AI 0, KO. # These nurnbers reflect the number of blots inspected visuatly (due to uneven running of the gel).

Fig. 4-4. Quantification of Complex III subunits in various Qcr7 mutant strains by densitometry. Panel 1: Histograms were constructed by averaging the density of each subunit from a number of blots (see table on panel I I ) . The density is expressed as a percentage of the respective subunit compared to the overexpressed wild type of each blot. a) Histogram representing quantitation for the 14 kDa and 1 1 kDa subunits. Error bars correspond to the standard error. Where error bars are not shown, the standard error is too small to be visible. b) Histogram representing quantitation for the mature ISP (m-ISP) and combined intermediate and mature ISP (al1 ISP). Error bars correspond to the standard error. Where error bars are not shown, the standard error is too small to be visible. c) Histograrn representing quantitation for mature cytochrome c, (m-cyt c,) and combined intermediate and mature cytochrorne c, (al1 cyt c,). d) Histogram representing quantitation for core protein 1 (core 1 ) and core protein 2 (core 2).

amphipathicity and eliminating these hydroxylated residues removes

some of the important features that are typical of mitochondrial

signal sequences. This could account for the lowered steady-state

levels of Qcr7 proteins seen in mitochondria isolated from strains

expressing the truncated versions of this subunit. Western blot

analyses of mitochondria which were treated with proteinase K

revealed that the mutant proteins were properly located inside the

mitochondria. It cannot be ruled out, however, that the observed

lowered levels of mutated Qcr7 proteins are due to degradation of

these proteins as a result of misfolding or a defect in assembly (see

"Future Directions" in Chapter 5).

Yeast overexpressing Qcr7p-al O do not contain any detectable

levels of the 14 kDa protein in the mitochondria although a band

migrating slower than the expected Qcr7 protein can be seen (Fig. 4-

3, lane 3). I t is unlikely that this band corresponds to an aberrantly

running Qcr7p-al O, however, since a faint band of the same size can

be visualized in lanes 6 and 7. In addition, blots performed with a

different antibody, which among other subunits also recognizes the

Qu7 protein, do not show the presence of this band. Hence, it may

be speculated that the Qcr7p-Al O is not imported into mitochondria

t o detectable levels because, in addition to its compromised

amphipathicity and elimination of hydroxylated residues Ser-4, Thr-

6, and Ser-7, it contains a negatively-charged aspartate as the

second residue from the N-terminus. If indeed the Qcr7 protein N-

terminus is involved in import, this residue might repel the protein

from the negatively-c harged phospholipid backbone of the membrane

and decrease the eff~ciency of import to levels below detection.

lmmunoblotting of the cytosolic protein fraction from the

mutant with QcrTp-Al O did not show an accumulation of Qcr7p-Al O

precursor in the cytosol (results not shown). This is not unexpected

as impon of rnitochondrial proteins is tightly coupled to synthesis

and it is rare that mitochondrial precursor proteins can be detected

in the cytoplasm (16% 164). Hence, Qcr7p-A10 is either imported

quickly after synthesis, or CO-translationally or, alternatively, it is

degraded rapidly due t o impaired import. Further experiments need

t o be performed to resolve the issue of whether Qcr7p-A~O is not

imported and degraded in the cytoplasm or whether it is degraded in

the mitochondria due t o an unassembled, partially assernbled, or

misfolded mutant protein.

The amino-terminus of the Qcr7 protein is essential for assembiy of

the bel complex

Examination of mutants with truncated proteins Qcr7p-A?,

Qcr7p-Al O, Qcr7p-A14, and Qcr7p-~20 with respect t o the levels of

the other subunits (refer to Figs. 4-3, 4-4, and 4-5) of complex III

indicates that the content of core protein 1 does not V a r y

significantly. Core protein 2 seems to be slightly reduced in al1

respiration-deficient mutants except in that with Qcr7p-

~7(RlOl/G12V). Intermediate ISP (i-ISP) whose bipartite signal

sequence has been processed once, is present a t lower arnounts in

the strain expressing the Qcr7p-~7 than in the strain with wild type

Qcr7 protein. The strain carrying Qcr7p-~14, however, contains only

trace amounts of this intermediate and i-ISP is undetectable in

strains with Qcr7p-a20 and Qcr7p-al0 and in YSM-qcr7A. Despite

the decreased leveis or absence of this intermediate, mature ISP is

present in al1 the mutant strains, showing decreased levels t o about

75% of wild type in mutants with Qcr7p-A14 and Qcr7p-~20. Yeast

expressing wild type Qcr7 protein and Qcr7p-A7 contain comparable

levels of mature ISP, whereas the remaining mutants have lower

amounts. The strain with Qcr7p-A? contains levels of 11 kDa

subunit comparable t o the wild type, the strains expressing Qcr7p-

A 14 and Qcr7p-A 2 0 have intermediate levels of this subunit

(approxirnately 60% of wild type), and the lowest levels of 11 kDa

subunit are seen in the strain expressing Qcr7pa l O and in YSM-

qcr7A. Examination of the levels of cytochrome cl suggests that

combined intermediate and mature cytochrome cl are reduced

approximately by 40% in the strain with Qcr7p-AI 4, and by over 60%

in strains with Qcr7p-A20 and Q c r 7 p - ~ l O and in YSM-qcr7A. In

summary, this initial analysis o f the truncated Qcr7 proteins

indicates that deletion of seven or more residues from the amino-

terminus of the Qcr7 protein leads to levels of this protein that are

reduced by approximately 55%; truncation of seven residues leads to

lowered levels of intermediate ISP and intermediate cytochrome cl.

In addition, truncation of 10 or more residues results in lowered

steady-state levels of the 11 kDa subunit, as well as intermediate

and mature iron-sulfur and cytochrome c l proteins. These results

implicate the amino-terminus of the Qcr7 protein in the functional

assembly of the bel complex (33.47,49, 55,60, 133.152).

Because the strain expressing Qcrfp-a7 was the least

compromised of the mutants in assembly of a functional complex III,

I decided to analyze the effect of point mutations in the context of a

A 7 deletion. The mutations targeted two charged residues, Arg-1 O

(mutated to Lys and Ile) and Asp-13 (mutated to Val). Three strains

were studied: a strain with Qcr7p-d7(RI OK), a strain with Qcr7p-

A7(D13V), and a strain containing Qcr7p-a7(R1 OVG12V). The

analysis presented in Chapter 3 indicated that strains expressing

Q c r 7 p - ~ 7 and Qcr7p-a7(RI OK) were respiration-deficient only a t

3 7OC. Cornplex Ill-linked activities measured in mitochondria from

these strains grown a t 300C were comparable t o the activities of

wild type cells. Strains expressing Qcr7p-A 7(Dl3V) and Qcr7p-

A7(R 1 0VG12V) were respiration-deficient at RT, 300C, and 370C.

W hereas the strain containing Qcr7p-A~(D 1 3V) lacked complex III-

linked enzyme activity, the strain with Qcrip-a7(R1 OVG12V)

contained a trace of complex Ill-linked activity.

Examination of the subunit composition of complex III in

mutant strains (Fig. 4-5) indicated that, of al1 the mutants studied

in detail, only the respiration-deficient mutant with Qcr7p-

87(D13V) contained levels of 14 kDa subunit comparable to that of

the strain overexpressing the wild type QCR7 gene. In strains

containing Qcr7p-d7(R1 OK) and Qcr?p-~7(RlOI/G12V) the amount of

14 kDa subunit is reduced to levels similar to that of yeast strains

expressing Qcr7p-~7, Qcr7p-~14, and Qcr7p-~20 (Figs. 4-3 and 4-4).

All the other subunits of the bc, complex in the yeast containing

Fig. 4-5. Western blot analyses of mitochondrial proteins from YSM-qcr7a strains overexpressing wild type Qcr7, Q c r 7 p - a 7 ( D 1 3 V ) , Qcr7p-A 7 ( R 1 OU), and Qcr7p- A 7 ( R 1 01/G 1 2V), respectively. Mitochondrial proteins (1 00pg) isolated from the above strains were dissolved in SDS-PAGE buffer containing DTT and heated for 3 min at 95oC. Samples were run on a 1 6% polyacrylamide gel and then transferred t o a nitrocellulose membrane. The blot containing core proteins 1 and 2, the ISP, the 11 kDa and 14 kDa subunits was probed with an antibody detecting al1 those subunits. The blot containing cytochrome c, was probed with a monoclonal antibody raised against this subunit. Lane 1: protein from YSM-qcr7A overexpressing wild type; lane 2: protein from YSM- q c r7A overexpressing R I OK; lane 3: protein from YSM-qcr7A overexpressing D l 3V; lane 4: protein from YSM-qcr7~ overexpressing R I OVG12V.

Qcr7p-~7(RlOK) are present in amounts comparable to the wild type.

Strains expressing Qcr7p-h7(D13V) and Qcr7p-~7(RlOI/G12V), have

core protein 1 levels comparable to the wild type, whereas

densitometry indicated that the level of core protein 2 is reduced in

the strain with Qcr7p-A i ( D l 3 V ) , similar as in the respiration-

deficient strains Qcr7p-A 1 O, Qcr7p-~14, Qcr7p-A20, and YSM-qcr7~

(Figs. 4-4 and 4-5). Mature ISP and cytochrome c, levels are sirnilar

to the wild type in yeast with Qcr7p-~7(Rl OVG1 ZV), but combined

intermediate and mature cytochrome c l and ISP are reduced by

approximately 30% in this mutant. The mutant containing Qcr7p-

~7(D13V) has lowered levels of both intermediate and mature ISP

and cytochrome c,. Although only mature ISP and cytochrome c,

contain catalytic activity, the reduced levels of their intermediates

are indicative of a higher turnover rate due t o an unassembled or

weakly assembled complex. As for the 11 kDa subunit, it is severely

reduced in the mutants with Qcr7p-h7(D13V) and Qcr7p-

a7(R1 OI/G12V) with the lower apparent level being in the latter.

Since an antibody was not available to monitor cytochrome b, I

assessed the presence of holo-cytochrome b in mitochondria by

spectrophotometric analyses (Fig. 4-6). Some samples could not be

assayed for cytochrome b content as they did not grow on non-

repressive carbon sources. Panel (a) in Figure 4-6 shows typical

spectra from the strain expressing the wild type QCR7 gene. In the

top part of panel (a) reduced minus oxidized spectra are seen

corresponding to cytochromes c ic , at 550 nm and cytochromes a+a,

a t 605 nm. In the bottom part of panel (a), reduced minus oxidized

spectra are seen corresponding to cytochrome b at 560 nm and

cytochromes a+a, at 605 nm. With respect to the other strains, the

difference spectra show that the strain carrying the conservatively

substituted Qcr7p-~7(R1 OK) is comparable t o that of the mutant

with Qcr7p-~7. The levels of holocytochrome b from these two

mutants are comparable t o the wild type, whereas the levels of

holocytochrome c+c, are somewhat reduced. In contrast, strains

expressing Qcr7p-~?(RlO!/G12V) and Qcr7p-~7(D13V) displayed

significantly lowered and undetectable amounts of holocytochrome

b, respectively, by this technique. The levels of holocytochrome c ic ,

were comparable in mutants with Qcr7p-A7 and Qcr7p-

~7(R101/G1 ZV), whereas the mutant with Qcr7p-b7(D13V) displayed

slightly reduced levels (Fig. 4-6).

The reduced levels of cytochrome b in strains expressing

Qcr7p-a7(RI OI/G12V) and Qcr7p-~7(D13V), correlate with the

decreased levels of overall ISP and cytochrome c,, as identified by

immunoblotting (Fig. 4-4). Although, as identif ied by

immunoblotting, mature cytochrome c, is comparable to the wild

type in the mutant with Qcr7p-b7(RlOI/G12V) and only slightly

reduced in the mutant with Qcr7p-b7(D13V), the lower relative

levels of holocytochrome c+c,, as seen by spectrophotometry, may be

due to decreased maturation of apocytochrome c, t o holocytochrome

c, as a result of an unstable or a weakly assembled complex. In

summary, immunoblotting and spectrophotometric analyses have

shown that combined intermediate and mature ISP, as well as

holocytochromes b and c+c, are reduced in the mutant with Qcr7p-

~7(D13V) when compared to the mutant with Qcr7p-~7(RlOI/G12V).

Keeping in mind that the mutant with Qcr7p-A7(D13V) has slightly

higher levels of 11 kDa subunit and significantly higher levels of

Qcr7 protein in the mitochondria than the mutant with Qcr7p-

a7(RlOI/G12V), and considering the results from the enzyme

activities (Chapter 3), the varying levels of complex III subunits

implicate a role for the Qcr7 protein amino-terminus in assembly of

a subcornplex/bc, complex.

Fig. 4-6. Difference spectra of the cytochromes. For spectral analyses of the cytochromes, mitochondria (2mg/mL) were resuspended in 0.1 M potassium phosphate (pH 7.4)-0.25 M sucrose- 0.5% cholic acid (33). TO obtain a spectrum containing cytochromes c+c, and cytochromes a i a , , a ferricyanide oxidized spectrum was subtracted from a dithionite reduced spectrum (top panel). To obtain a spectrum containing cytochrome b only, dithionite reduced minus ascorbate-0.2 mM TMPD reduced samples were run (bottom panel). Spectra were recorded on a DW-Za Aminco spectrophotometer from 520 to 620 nm.

cl- -/--

Co-immunoprecipita tlons

Since the N-terminus of the Qcr7 protein seemed to play a role

in assembly of the bel complex, it was of interest t o determine

which subunits the N-terminus associates with and in which order.

These issues were approached by attempting t o CO-precipitate the

subunit/s surrounding the 14 kDa protein. lmmunoprecipitations

were carried out under a variety of conditions using an anti-Qcr7p

antibody raised in chicken and in rabbit to a carboxy-terminal

peptide of the protein. The immune response is unique for each

animal, hence the two anti-Qcr7 antibodies obtained varied with

regard to their specificities. The antibody raised in chicken did not

immunoprecipitate the Qcr7 protein, hence the anti-Qcr7p antibody

raised in rabbit was employed for all imrnunoprecipitation

procedures. The results proved t o be inconclusive (results are not

shown). Three secondary antibodies were used in Western blot

analyses t o determine which complex III subunits had CO-

precipitated with the 14 kDa protein. The first one, raised in rabbit,

recognizes core proteins 1 and 2 , the ISP, and subunits 7 and 8; the

other two were monoclonal antibodies raised in mice against the ISP

and cytochrome c I , respectively. When the rabbit antibody was

employed to detect immunoprecipitated proteins on the immunoblot

there was a significant amount of cross-reaction with the anti-

Qcr7p antibody since both were raised in the same animal species.

Hence, the large molecular weight core proteins could not be

detected due to interference from the IgG heavy chains (about 5 0 kDa

in size). Even under non-reducing conditions there was sufficient

background on the immunoblot corresponding to the heavy chains to

obscure the potentially CO-precipitated core proteins. Similarly, the

ISP could not be detected with this antibody due to interference

from the IgG light chains (approximately 25 kDa in size). However,

when ernploying the mouse monoclonal anti-cytochrome c l and anti-

ISP antibodies, there was minimal interference frorn the IgG (H+L)

chains of the anti-Qcr7 antibody used in the immunoprecipitation.

Blots visualized with these secondary antibodies seemed to indicate

that cytochrome cl and the ISP did not CO-precipitate. This notion

was substantiated by the identical band profile seen in the two

immunoblots. That is, since the bands that could be seen in the two

blots were identical, they could not correspond to cytochrome cl and

the iron-sulfur protein, but rather to the minimal interference of

the IgG light chains which migrate a t approximately the same rate

as cytochrome cl and the ISP.

Under some conditions employed, a protein band that migrates

in the region of the 11 kDa protein could be visualized in the wild

type, but not in the strain YSM-qcr7~. On the other hand, under

different conditions there appeared to be such a protein band even in

the disruption strain suggesting that the protein migrating in the

region of 11 kDa seen following immunoprecipitation is not the 11

kDa protein of the bci complex.

Results from immunoprecipitations following cross-linking

were inconclusive as well. No matter which of the three antibodies

described above were used to detect the proteins on the

immunoblots, the profile seen for al1 the mutant strains tested and

the wild type (whether under reducing or non-reducing conditions)

was the same. That is, the same band pattern was seen for al1 the

mutant strains, including YSM-qcr7A, and for the wild type. Since

the immunoprecipitations were carried out with an anti-Qcr7

antibody, this indicates that the observed bands are not relevant to

subunits from complex III. A new antibody raised against the 14 kDa

subunit or the 11 kDa subunit could perhaps be tested with better

results in the future (see "Future Directions", Chapter 5).

Deletion of seven residues from the amino-terminus does not impair

pro ton pumping

1 found that truncation of the amino-terminal seven amino

acids did not impair electron transport (that is complex Ill-linked

enzyme activity was not affected), hence I tested whether this

segment might contribute t o proton translocation. Generation of

ATP can be used as an indirect measure of the proton gradient. With

succinate as substrate, ATP synthesis in mitochondria is a measure

of the integrity of the proton gradient that is established by

complexes III and III+IV. When ferricyanide is used as electron

acceptor with succinate as substrate, the ATP produced is solely due

to the electrochemical gradient generated by complex III. As already

mentioned, to establish a proton gradient electron transport has to

be intact; hence, as expected, of al1 the strains containing truncated

Qcr7 proteins that were tested, only Qcr7p-~7 produced ATP in the

assay system. The amount of ATP produced in mitochondria from the

A 7 protein-containing mutant, however, was no different from that

in mitochondria from the wild type irrespective of whether the ATP

was generated by the activity of complex Ili alone or complexes

III+IV. Similarly, mutant strains expressing Qcr7 proteins with

residue substitutions in Ser-4 and Thr-6 did not show a decrease in

the amount of ATP produced when compared to the wild type (Table

4-1). As the yeast Qcr7 protein has a longer N-terminal extension

than its beef heart homologue, Qcr7 proteins with N-terminal

truncations of 14 and 20 residues (after Met-1) were synthesized to

test for the amount of ATP synthesized. Unfortunately, mutants

expressing Qcr7p-AI 4 and Qcr7p-A 20 completely lack complex III-

linked enzyme activities (see Table 3-2) and could therefore not

produce any ATP.

The strain expressing Qcr7p-d 7 is temperature sensitive

The growth of the yeast strain expressing the Qcr7p-~7 a t

370C (Fig. 4-7) displays a different profile from the phenotype a t

30oC (refer to Fig. 4-3). At 370C this strain is a pe t - mutant with

undetectable NADH-cytochrome c reductase activity. In this case

immunoblotting (Fig. 4-7) showed that the A 7 protein was not

present in the mitochondria (lane 1) although it was readily

detectable in YSM-qcr7~ overexpressing the wild type Qcr7 protein

(lane 2). This result may implicate involvement of the N-terminus

in mitochondrial targeting and import in a temperature sensitive

manner. Alternatively, the absence of Qcr7p-A7 in the mitochondria

rnay simply be the result of an intrinsically unstable protein and a

higher rate of degradation a t 370 C.

Table4-1: ATPS

Mutation

episomal wild type

~7

S4G

S4Y/11 1 V

S4G

S4WE109G

P2T/S4L

T6P

T6R

nt hesis

Complex III+IV Complex III

(- Ferricyanide) (+ Ferricyanide)

41 9 (8) +/- 73 1 12 (8 ) +/- 27

374 (2) 107 (2)

366 - 516 - 554 - 540 - 412 ( 2 ) 122

- 85

- 85 v

synthesized per milligram of protein (nmol of ATP/mg of protein) in 45 minutes in the presence or absence of femcyanide (for details see Material and Methods).

II

The numbers correspond to the total arnount of ATP

Fig. 4-7. Wes te rn b lo t analysis p e r f o r m e d w i t h mitochondrial membranes prepared f rom Y S M - q c r 7 ~ (g r o w n a t 370C) overexpressing Q c r 7 p - ~ 7 and the wild type Qcr7 protein. Mitochondrial proteins (1 00 pg, from cells grown a t 370C) were dissolved in SDS-PAGE buffer and heated for 3 min a t 950C. Samples were run on a 16% polyacrylamide gel and then transferred to a nitrocellulose membrane. The blot was probed with a polyclonal antibody that recognizes core proteins 1 and 2, the iron-sulfur protein, the 14 kDa and 1 1 kDa subunits. Lane 1: protein from the strain Y S M - q c r 7 ~ overexpressing Qcr7p-A7; iane 2 : protein from strain YSM-qcr7~ overexpressing the wild type Qcr7 protein.

4.4. DISCUSSION

From the analyses of the mutant strains presented in Chapters

3 and 4 it is evident that despite the 5 0 mutations that were

introduced into the QCR7 gene, only few caused a respiration

deficiency. Of 28 full-length Qcr7 proteins containing point

mutations that were examined, none caused any deficiencies a t RT,

30°C, or 37OC when expressed in the strain containing the qcr7 gene

disruption, YSM-qcr7A. On the other hand, some o f the mutant

strains expressing Qcr7 proteins with point mutations in the context

of a a7 deletion were respiration-deficient even though the strain

expressing Qcr7p-~7 by itself did not display such a defect at 30°C.

These results rnay indicate that while individual residues alone are

not crucial for the function of the Qcr7 protein amino-terminus, the

destabilized, but still functional Qcr7p-~7 can be disruptive with

respect t o cornplex III function when additional residues are

mutated. This theory would explain why substitution of residues

without an added deletion, as in the mutants containing full-length

proteins, did not result in a deficiency. Furthermore, it explains

why the strain expressing Qcr7p-A7 displays a phenotype equivalent

t o the wild type at 30°C, whereas some of the strains expressing

point mutations in the context of a ~7 deletion are pet-. A deletion

of seven residues rnay be tolerated, but any additional unconserved

substitutions in critical reg ions apparently exceed the threshold. If,

for example, the Qcr7 protein N-terminus is involved in assembly,

truncating this region could weaken the interaction with the other

subunith. Hence, truncation of the N-terminal seven amino acids

still allows assembly, but the complex is less stable. If, then,

additional deletions or point mutations are introduced into the N-

terminal region of the Qcr7p-A7, the secondary structure of the

protein may be further altered especially if the amino acid

substitutions are not conservative. This could cause the already

weakened interactions t o be disrupted. Construction of mutants

with full-length Qcr7 proteins showing normal activity, particularly

Qcr7pG12V and Qcr7pD13V, would strengthen this argument.

The results outlined in this chapter are consistent with this

model. The strain expressing Qcr7p-a7(R1 OK) with the conservative

amino acid substitution R l O K displays a phenotype identical t o the

strain containing the Qcr7p-A7; however, the strain containing the

protein with the non-conservative substitution, D l 3V, is a pe t -

mutant. The hypothesis that the preservation of the entire N-

terminal region and i ts inherent conformation are crucial rather

than the individual residues alone, is further substantiated by

strains expressing Qcr7p-A ï ( R l OT/K44N) and Qcr7p-

A ~ ( A ~ V / R I OT/Y14N/N53D) (see Chapter 3). The former of these

mutants is respiration-deficient, whereas the latter has a phenotype

comparable t o strains expressing Qcr7p-a7 and Qcr7p-a7(RlOK).

This observation rnay argue against a functional involvernent of Arg-

10, but on the other hand, one has t o be cautious in drawing

conclusions from the strain with the quadruply mutated Qcr7p-

a7(A9V/R1 OT/Y14N/N53D), as the substitution of Arg-1 O by

threonine could potentially be compensated for by the other amino

acid substitutions. The wild type phenotype of the strains

expressing full-length Qcr7p-R 1 OT, Qcr7p-R 1 01, Qcr7p-D13K, and

Qcr7p-Dl SE further confirm the notion that preservation of the N-

terminus and its inherent conformation are important. Since,

however, full-length proteins Qcr7p-K44N, Qcr7p-G1 ZV, and Qcr7p-

Dl 3V were not constructed, it cannot be ruled out cornpletely that

these individual residues are essential. This seems improbable

however, since none of the strains expressing full-length Qcr7

proteins with point mutations caused a respiratory chain defect, no

matter whether the charged residues were substituted for by a polar

residue (RlOT), a residue of opposite charge (Dl 3K), or a hydrophobic

residue ( R I 01). Furthermore, the mutant containing the truncated

protein Qcr7p-~7(N53S/EI 1 6G) is pet-, whereas expression of

neither of the full-length proteins Qcr7p-N53S and Qcr7p-N53D

resulted in a respiration-deficient phenotype. In this case, the pet-

phenotype cannot be attributed to the mutation at residue Glu-1 16,

since Hemrika e t al. (49) have truncated a region including this

residue and found that the mutant containing this protein retains

approximately 40% of complex Ill-linked activity and is not

respiration-deficient. Hence, the p e t - phenotype of the strain

expressing Qcr7p-~7(D13V) is probably the combined result of an

unconserved substitution of a critical residue, and the N-terminal

truncation of seven residues.

Recent results obtained from preliminary growth analyses of

newly generated mutants indicate the following: al1 mutants

containing Qcr7 proteins with residue substitutions in the context

of the truncated protein Qcr7p-A7 are respiration-deficient. These

include mutants with proteins Qcr7p-~?(D13Y), -(Dl 3A), -(Dl 3G),

-(Dl 3N), -(G12V), - (R I OI), and -(A9V/G12E). In contrast, the only

new mutant containing a protein with a residue substitution in the

context of the full-length protein, Qcr7pG1 ZV, did not display a

pe t - phenotype. This confirms the prediction that truncation of

seven residues from the amino-terminus is dominant and that any

additional, non-consewative residue substitutions of important

amino acids are disruptive. These results also make it less likely

that a full-length Qcr7p-Di 3V would cause a respiratory chain

defect (So-Young Lee, personal communications).

Co-precipitation of proteins was atternpted by employing an

anti-Qcr7 antibody in order to reveal the identity of any subunit

cornplexed with the 14 kDa protein. Although the antibody used was

capable of immunoprecipitation, under conditions where subunits

were not cross-linked, CO-precipitation could not be achieved. It is

thus probable that the anti-Qcr7 protein antibody cannot access the

14 kDa subunit without disrupting the interactions with the

surrounding subunits. Hence, the yield of only the 14 kDa subunit

following immunoprecipitation. This speculation is in agreement

with previous studies which suggested that the 14 kDa subunit is

located in the interior of the enzyme complex (66) .

CHAPTER 5

Discussion and

Future Directions

CHAPTER 5

Discussion

Previous inactivation of the QCR7 gene has shown that the

Qcr7 protein is an essential component of ubiquinol-cytochrome c

oxidoreductase (33). The disrupted strain displays a pet- phenotype

and does not grow on non-fermentable carbon sources. Previous

studies have implicated the Qcr7 protein in the functional assembly

of ubiquinol-cytochrome c reductase (33 , 4% 55, 60-63). The N-

terminus of the Qcr7 protein is believed to be oriented towards the

matrix in yeast (66 , 130) and in its homologue of beef heart

mitochondria (*3# 31 1. Because proteolysis of the N-terminal seven

amino acids of the beef heart mitochondrial counterpart leads to a

decreased H+/e- ratio, Cocco e t al. (131 ) suggested that this subunit is

involved in proton uptake from the rnatrix with subsequent transfer

of these protons t o the hypothetical ubiquinone binding pocket at

center N. Since, however, proteolysis also resulted in a small

amount of cleavage from the Rieske ISP, it cannot be ruled out that

the ISP is in fact responsible for the decreased H+/e- ratio.

In the current work, the role o f the Qcr7 protein amino-

terminus was investigated with respect to three putative functions;

(1) proton translocation, (2) mitochondrial targeting, and (3)

assembly of the bcl complex. Although the Qcr7 protein does not

contain a cleavable N-terminal targeting sequence, the Qcr7 protein

is unlikely to follow the import pathway used by mitochondrial

proteins without cleavable N-terminal leader sequences, such as the

ADP/ATP carrier (143). In addition, the N-terminal amino acids

display features characteristic of signal sequences, such as net

positive charge, and a number of hydroxylated and hydrophobic

residues. The N-terminus o f the beef heart mitochondrial

counterpart has been postulated to form an amphipathic a-helix (231,

a feature that is typical of mitochondrial targeting sequences. The

above issues were approached by inactivating the QCR7 gene and

complernenting the resulting mutant strain, YSM-qcr7~, with a

number of qcr7 genes containing point and deletion mutations. In

addition, the secondary structures of selected amino-terminal

peptides were studied by CD spectroscopy and by cornparison to their

beef heart homologues.

Circular dichroism studies of a 23 residue peptide from the

amino-terminus of the Qcr7 protein (peptide 4) indicated that this

region displays a-helical spectra in SDS and in methanol (Fig. 4-1).

The extent of a-helix formation of this peptide was comparable to

that of beef heart peptide 1 (residues 2-1 6, results not shown) and

similar t o beef heart peptide 2 (residues 2-26). These results

indicate that even though the primary sequences of the yeast and

beef heart N-termini do not show significant similarity, their

secondary structures are nevertheless similar. In addition, these

findings rnay point towards the importance of the conformation of

the N-terminus as a whole. Helical wheel plots o f the wild type N-

terminus show that the predicted a-helix is amphipathic in nature

(Fig. 4-2) in agreement with one of the typical features of

mitochondrial signal sequences.

Overexpression o f the genes encoding Qcr7p-87 and Qcr7p-

~ 7 ( R 1 OK) in YSM-qcr7~ restored complex Ill-linked enzyme

activities t o levels comparable t o the strain overexpressing the

wild type QCR7 gene a t 30°C. However, overexpressing the genes

encoding Qcr7p-A 1 O, Qcr7p-A14, Qcr7p-~20, Qcr?p-~7(RlOl/G1 ZV),

and Qcr7p-~7(D13V) did no t restore a respiration-competent

phenotype. This prompted me t o investigate whether the truncated

proteins are in fact transported into mitochondria. From Figs. 4-3,

4-4, and 4-5 it is evident that al1 the above mutated proteins with

the exception o f Qcr7p-A~O and Qcr7p-a7(D13V) are present in the

mitochondria a t levels comparable t o the functional Q c r 7 p - ~ 7 .

Althoug h these steady-state levels are signif icantly reduced in

cornparison t o the wild t ype Qcr7 protein in the strain

overexpressing QCR7, the levels are nonetheless comparable t o those

of Qcr7 protein in the parental strain W303-1 B.

Respiration-deficient mutants with Qcr7p-Al0 and Qcr7p

~ 7 ( D 1 3 V ) displayed a different profile from the other Qcr7 proteins

(Figs. 4-3, 4-4, and 4-5). The strain containing Qcr7p-~7(D13V) is

the only mutant with Qcr7 protein steady-state levels comparable t o

the strain overexpressing the wild type QCR7 gene. One can argue

that the elimination of the negatively-charged Asp-13, an untypical

residue for rnitochondrial signal sequences, compensates for the

truncation of the seven N-terminal residues and restores protein

levels back t o those comparable to the strain overexpressing the

QCR7 gene. However, one cannot eliminate the possibility that this

protein is present at wild type levels because it is less susceptible

t o proteolysis in the mitochondria than the other truncated Qcr7

proteins. In contrast t o Qcr7p-A 7(Dl3V), Qcr7p-Al O is not

detectable in the mitochondria. Western blotting of the cytosolic

protein fraction from the strain with Qcr7p-Al O did not show an

accumulation of this subunit in the cytosol. Hence, it is not clear

whether the absence of detectable Qcr7p-Al0 is the result of an

unstable protein which is degraded in the cytoplasm or the

mitochondria t o levels below detection, or whether Q c r 7 p - ~ l O is

degraded in the cytoplasm because it is not imported. I t is

conceivable, that Qcr7p-al O is not imported into mitochondria due

to the negative charge of Asp-13 which, in this truncated protein, is

exposed a t the amino-terminus and might repel the protein from the

negatively charged phopholipid backbone. Negatively charged

residues are uncommon in mitochondrial signal sequences ( iso l '531,

but they may not always be as detrimental to the irnport process

depending on their location or orientation. Further experiments are

clearly needed to resolve the issue between import and degradation

(see "Future Directions").

If the Qcr7 protein was to follow the same import pathway as

proteins with a cleavable amino-terminal signal sequence, then

deletion of the amino-terminus could conceivably result in

decreased import (1 25). For example, truncated proteins may not

interact with cytosolic chaperones as efficiently or, alternatively,

they rnay not bind as tightly to the OMM receptors. The absence of

Qcr7p-~7 at 370C could thus be due to a higher degradation rate at

that temperature resulting from the impaired binding t o cytosolic

chaperones. Roise e t ai. ( 1 5 4 ) have suggested that part of the

mechanism of protein import is the perturbation of the phospholipid

bilayer by the surface active amphipathic helix of the presequence.

This ability of the presequence t o cause a local defect in the

membranes would then create a route for the rest of the protein t o

follow. It is thus conceivable that the truncated proteins of this

study cannot enter the membrane as efficiently, since their

amphipathic character and inherent lytic properties have been

decreased by shortening the presequence. Presequences must be able

t o form amphipathic structures with the charged and hydrophilic

residues located on one side of the helix and the hydrophobic

residues on the other side ( 1 W 155). As indicated by the CD spectra,

the wild type N-terminus is capable of forming an a-helix in a

membrane-mimetic environment (Fig. 4-1 ). In addition, helical

wheel plots of the wild type N-terminus show that this helix is

amphipathic. In contrast, helical wheel plots of Qcr7p-A7 (Fig. 4-2)

and Qcr7pdl O, Qcr7p-AI 4, and Qcr7pA20 (not shown) show that the

amphipathicity o f these helices, if indeed they form helices, is

compromised. Baker and Schatz (95) have shown that 25% of

randomly generated peptides are capable of targeting proteins into

mitochondria, although a t lower efficiency than authentic signal

sequences. Hence, if the Qcr7 protein amino-terminus does in fact

play a role in mitochondrial import, then elimination of some of the

features typical of mitochondrial signal sequences, as seen here in

the truncated Qcr7 proteins, does not have to prevent irnport into

mitochondria, but merely reduce its efficiency. To make a definite

statement on the efficiency of mitochondrial import, further

experiments can be performed (see "Future Directions"). Such

experirnents were not atternpted as a part of this study, as the

question whether the Qcr7 protein amino-terminus is essential for

import into mitochondria was answered by the presence of Qcr7p-

a20 in the mitochondria.

Since transformation of the gene encoding Qcr7p-A7 into the

strain YSM-qcr7a restored complex Ill-linked enzyme activities to

levels comparable to the strain expressing the episomal wild type

Qcr7 protein a t 300C (Table 3-Z), 1 proceeded to test whether the N-

terminal seven amino acids play a role in proton transfer by

assaying the amount of ATP synthesized in mitochondria. As ATP

synthesis is driven by the protonmotive force, the amount of ATP

produced can be used as an indirect assessment of the integrity of

the proton gradient. I found that concentrations of ATP produced

were comparable in strains expressing wild type Qcr7 protein,

Qcr7p-A7, Qcr7p-S4G, Qcr7p-S4Y/IIlV, Qcr7p-S4Y, Qcr7p-

S4G/E109G, Qcr7p-P2T/S4L, Qcr7p-T6P, and Qcr7p-T6R (Table 4-1 ).

From this it can be concluded that in yeast, the involvement of the

N-terminal seven amino acids in proton translocation and ATP

synthesis is unlikely to be critical. Unfortunately, it was not

possible to test the N-terminal region in yeast that is relevant to

the region found to be critical in the beef h e m homologue, as yeast

with truncated proteins Qcr7p-a14 and Qcr7p-a20 did not contain

any complex Ill-linked activitieç. Although these findings may seem

to contradict the studies performed in beef heart mitochondria (1 311,

one has to remember that the yeast homologue contains a longer N-

terminal extension. Furthermore, one has to keep in mind that Cocco

et al. truncated the 13.4 kDa subunit (and others) by subjecting the

already assembled complex t o proteases, thereby circumventing a

potential assembly problem. Hence, it cannot be ruled out that the

region between residues 9 and 21 of the yeast Qcr7 protein are in

fact involved in proton translocation.

Although the N-terminal seven amino acids (after Met-1) of

subunit 7 may facilitate import into mitochondria or confer

stability to the complex, the results of this work suggest that the

amino-terminus of the Qcr7 protein, especially the region between

residues 8 and 20 (after Met-1), is essential for the formation of a

functional bc, complex. Steady-state levels of the majority of

truncated Qcr7 proteins are reduced when compared to the

overexpressed wild type (Fig. 4-4), but the pet- phenotype of the

respiration-deficient mutants cannot be attributed to these reduced

levels as they are comparable to the functional Qcr7pa7. Hence, the

p e t - character of the strains expressing Qcr7 proteins with

truncations (and point mutations) is more likely to be the result of

the decreased levels of some of the other subunits of complex III.

lmmunoblotting shows that the steady-state level of the 11

kDa subunit is decreased in al1 respiration-deficient mutants.

Combined intermediate and mature cytochrome c, and ISP are also

decreased in al1 respiration-deficient mutants tested except for the

mutant with Qcr7p-~7(RlOI/G12V); this mutant contains wild type

levels of mature cytochrome c, and ISP, but lowered overall levels of

these proteins. In addition, spectrophotometric analyses of the

respiration-deficient strain containing Qcr7p-a7(D13V) show that

holocytochrome cl is sig nificantly decreased in this mutant when

compared to the wild type, in agreement with the observations from

the immunoblotting. Furthermore, holocytochrome cl seems to be

reduced slightly in the respiration-deficient mutant with Qcr7p-

~7(R101/G12V) and the respiration-competent mutants with Qcr7p-

A7 and Qcr7p-a7(Rl OK). Nonetheless, the strain with Qcr7p-A7,

which has complex Ill-linked activities indistinguishable from the

wild type a t 30°C, contains lowered intermediate cytochrome cl and

ISP.

Although the N-terminal seven residues of the Qcr7 protein are

not essential for assembly, the complex seems to be less stable

without them. This is indicated by the reduced intermediates of

cytochrome c, and ISP which are the result of a higher turnover rate

of the complex in the mutant containing Qcr7pWA7. When comparing

mutants with Qcr7p-A7, Qcr7pa14, and Qcr7p-A20, one can see that

with increasing N-terminal truncation the levels of intermediate

cytochrome c, and ISP decrease to be virtually absent in the mutant

containing Q c r 7 p - ~ 2 0 . This makes sense since the larger

truncations presumably result in more compromised assembly and

since processing of intermediate ISP into mature ISP is predicted

not to occur until after insertion of this protein into the assembled

complex (133 . 156) . Hence, even though mature ISP and mature

cytochrome c, are present, the reduced levels of these proteins as

seen in some mutants and the absence of their intermediate forms

are a typical indication of an assembly defect (33, 47, 49. 55. 60. l33 ,

152). For example, mature ISP is reduced and intermediate ISP is

undetectable in the study performed by Schoppink e t al. (331, which

describes the previous inactivation of the 14 kDa subunit and in

which the authors conclude that the obsewed defect is due to

compromised assembly of the complex. This profile is similar t o the

profiles seen for YSM-qcr7~, and the strains expressing Qcr7p-~10,

and Qcr7p-~20, respectively. In addition, in the study by Hemrika e t

al. (55) who have concluded that the aromatic nature of residue 66 of

the 11 kDa subunit is important for assembly o f a functional

complex III, intermediate and mature ISP are characteristically

reduced in a mutant in which residues 66 to 70 (YWYWW) have been

replaced by the sequence SASAA. A revertant of the SASAA mutant

with growth rates approximately equal to the wild type was

sequenced and found to contain the sequence FASAA. Analysis of

this revertant revealed that mature ISP is present a t comparable

levels t o the wild type, whereas intermediate ISP is reduced. This

profile is similar to the profile seen for the ISP in the mutant with

Qcr7p-~7. Furthermore, in the study in which Hemrika e t al. (49) have

shown that the C-terminus of the 14 kDa subunit is involved in

assembly of a functional enzyme, intermediate and mature ISP are

characteristically reduced. Also, a study by Crivellone e t al. (60) has

shown that intermediate and mature ISP are reduced in al1 mutants

in which the genes encoding complex III subunits have been deleted

and, as a result, the complex does not assemble into a functional

enzyme. Thus, numerous mutational studies of complex III subunits

have been performed, where reduction of intermediate and mature

(or interrnediate only) forms of ISP and cytochrome c, have been

interpreted as being indicative of an impairment of the assembly

process. The levels of ISP and cytochrome c,, as documented by

previous studies and by this study, faIl between the wild type levels

and the drastically lowered levels seen in strains in which the QCR7

gene has been deleted. However, in spite of the interpretation of

lack of intermediate ISP and cytochrome c, proteins in complex III

mutant analysis as being indicative of lack of complex III assembly,

there is a paucity of experimental work in the literature to

substantiate this assurnption.

The hypothesis that truncation o f the N-terminal seven

residues (of the mature protein) destabilizes the assembly of the bc,

complex, but that any further truncation or non-conservative

substitution of important residues is detrimental t o the assembly

process is substantiated by the following observations: i) truncation

of seven residues from the amino-terminus by itself results in

lowered levels of cytochrome c, and ISP intermediates; ii) any

truncation beyond seven residues of the Qcr7 protein amino-

terminus results in lowered levels of intermediate and mature

cytochrome c, and ISP, as well as the 11 kDa subunit, despite levels

of 14 kDa subunit comparable to the strain with Qcr7p-A7; iii) the

substitution of valine for Asp-13 in the context of a A 7 truncation

results in severely reduced levels of the 11 kDa subunit,

intermediate cytochrome c, and ISP, mature ISP, holocytochrome c,

and holocytochrome b; the mutant containing Qcr7p-~7(RlOI/G12V)

has severely reduced levels of the 11 kDa subunit, reduced levels of

intermediate cytochrome c , and ISP, as well as reduced

holocytochrome b and holocytochrome c,; iv) none of the mutants

with full-length Qcr7 proteins containing only point mutations

display either a respiratory chain defect or lowered levels o f

intermediate or mature cytochrome c, and ISP (not shown). This is

the case, no matter whether the charged residues were substituted

for by a polar residue ( R I OT), a residue of opposite charge (Dl 3K), or

a hydrophobic residue (RI 01). Furthermore, the mutant containing

the truncated protein Qcr7p-~7(N53S/E116G) is pet- , whereas

expression of neither of the full-length proteins Qcr7p453S and

Qcr7p-N53D resulted in a respiration-deficient phenotype. In this

case, the pet- phenotype is less likely due t o the mutation a t residue

Glu4 16, since Hemrika e t al. (49) have truncated a region including

this residue and found that the mutant containing this protein

retains approximately 40% of complex III-linked activity and is not

respiration-deficient. Hence, the evidence from al1 the mutants

constructed in the context of Qcr7p-A7 and the new finding that the

mutant with the full-length Qcr7p-G1ZV is comparable t o the wild

type according to growth, speaks for the dominant effect of the N-

terminal truncation of seven residues.

The observed decrease in the levels of complex III subunits

other than the Qu7 protein cannot be explained by an unstable Qcr7

protein which is misfolded and subsequently degraded. In addition,

the presence of most mutated Qcr7 proteins (except Qcr7p-al O and

Qcr7p-~7(D13V)) in the mitochondria at comparable levels which are

also similar to, or higher than the level of Qcr7 protein in the

parental strain W303-1 B, speaks against their lowered levels as a

result of degradation. If the mutated Qcr7 proteins were unstable,

one would expect them to be degraded to levels below detection or

t o varying levels as shown in the study by Crivellone e t al. (60 ) . In

addition, the fact that the majority of mutated Qcr7 proteins are

present in the mitochondria a t comparable levels, despite their

different mutations, speaks against their instability as a result of

mutation. The fact that so many mutants with wild type phenotype

(Table 3-2) could be isolated speaks for the stability of the protein

and indicates that overall configuration is perhaps more important

than the individual residues. A precedent of this type of phenornenon

is seen in some of the subunits of complex I from a variety of

eukaryotes where few amino acid residues are consewed (18).

Nevertheless, the comparable levels of Qcr7 protein seen in

most mutants by themselves do not rule out the possibility that this

protein is partially degraded and that the assembly defect of

complex III follows from that. However, the levels of the other

subunits of the complex speak against an assembly defect as a

result of a misfolded Qcr7 protein. When comparing the degree of

assembly t o that in the mutant strain YSM-qcr7A, in which a Qcr7

protein is not synthesized, the following observations speak for an

increase in the assembly of a subcomplex or cornplex III itself (Fig.

4-4): i) elevated levels of 11 kDa subunit in al1 mutants when

compared t o YSM-qcr7a; ii) elevated levels of mature ISP in the

respiration-deficient mutants containing Qcr7p-~7(D13V), Qcr7p-

A14, and Q c r 7 p ~ 2 0 when compared t o YSM-qcr7A; iii) elevated

levels of mature cytochrome c, in some of the respiration-deficient

mutants when compared to YSM-qcr7A. Taken together, these results

speak against a mechanism involving total misfolding followed by

degradation independent of the other subunits of the complex, as

this would give levels of 11 kDa subunit, ISP and cytochrome c,

identicai to those seen in YSM-qcr7A.

One could possibly argue that truncation of the Qcr7 protein N-

terminus alters the state of acetylation of the truncated proteins as

opposed to the wild type and that this could influence the assembly

process. This seems unlikely, however. According to Persson e t a/.

(167) Ser and Ala are the most common residues at position 1 in

acetylated proteins, followed by Met. Furthermore, Asp, Glu, Ser,

and Thr are common a t position 2 and Lys is generally

overrepresented in the N-terminal 10 residues of the 250 acetylated

proteins that were characterized. When applying these guidelines to

the wild type and truncated Qcr7 proteins, the wild type Qcr7

protein and Qcr7p-A14 are not candidates for acetylation; Qcr7p-~7,

410, and - A 2 0 could potentially be acetylated, with Qcr7p-A7 being

the most likely candidate. Since Qcr7p-~7 is similarly functional in

assembly to the wild type whereas Qcr7p-AI 4 and Qcr7p-A20 are

similarly disruptive with respect to assembly, it is highly

improbable that the potentially different states of acetylation are

responsible for the observed assembly defect. In addition, it is not

clear whether the Qcr7 protein is acetylated in the first place as

mitochondrial proteins are less frequently acetylated than their

non-mitochondrial isozyme counterparts. This makes sense since

acetylation is dependent on the availability of acetyl Co-A which in

turn is decreased when the tricarboxylic acid cycle is active or

when yeast are grown under aerobic conditions (169).

Now, that the implication of the Qcr7 protein N-terminus in

assembly has been established, a model is proposed on the basis of

these findings which is compatible with the observations. Consider

the results obtained for the 11 kDa subunit: it is present a t wild

type levels in the mutant expressing Qcr7pd7, a t intermediate

levels in mutants containing Qcr7p-AI 4 and Qcr7p-AZO, but this

subunit is drastically lowered in strains with Qcr7p-a7(R101/GlZV)

and Qcr7p-~7(D13V). This observation suggests that the putative

interaction between the Qcr7 protein and the 11 kDa subunit occurs

beyond residue 8. In light of the proposed formation of a subcomplex

between cytochrome b, the Qcr7 protein, and the 11 kDa subunit (339

4% 559 60-631, this may suggest that the potentially destabilized

complex that is seen for the mutant containing Qcr7pa7, is the

result of a disruption of the putative interaction between

cytochrome b and the 14 kDa subunit. Examination of the data

obtained for the mutant containing Qcr7pd7(D13V) confirms the

above model. In this mutant the levels of 11 kDa subunit are

significantly lowered which can be explained by the substitution of

Val-13 for Asp-13 which could lower the extent of the putative

interaction of the Qcr7 protein and the 11 kDa subunit. Because of

the potential repulsion between the Qcr7p-~7(D13V) and the 1 1 kDa

subunit in addition t o the potentially destabilized interaction

between the Qcr7p-~7(D13V) protein and cytochrome b as a result of

the N-terminal truncation, further assembly into a multimeric

complex III is presumably unstable and the degree of assembly is

therefore decreased. This notion is conf irmed by the

spectrophotometric analyses of holocytochrome b, which indicate

that cytochrorne b maturation does not occur to detectable levels in

this mutant, and by the lowered levels of total apocytochrome cl,

holocytochrome cl, and mature ISP. Now consider the data obtained

for the mutant containing the Qcr7p-~7(RlOI/G12V) in light of the

postulated hypothesis. Of al1 mutants with detectable Qcr7 proteins

in the mitochondria, this mutant has the lowest levels of 11 kDa

subunit, but has intermediate levels of cytochrome b. The more

severe putative inhibitory effect between Qcr7p-~7(RlOl/GlZV) and

the 1 1 kDa subunit, as compared to that between Qcr7p-~7(D13V)

and the 11 kDa subunit, may be explained by a "double" severe

negative effect due to the creation of two hydrophobic residues. The

higher level o f holocytochrome b seen in the mutant with Qcr7p-

~7(R101/G12V) as opposed to that with Qcr7p-~7(D13V), might be

explained by a closer proximity of Arg-1 O tu cytochrome b than Asp-

13. Hence, in this mutant, as opposed to the mutant with Qcr7p-

~7(D13V), the newly created lie-1 0 (and perhaps Val-1 2 ) may repel

the 11 kDa subunit, but, on the other hand, may strengthen the

putative hydrophobic interaction with cytochrome b. This more

stable interaction with cytochrome b in the mutant containing

Qcr7p-d7(Rl OI/G1 ZV), as opposed to the mutant containing Qcr7p

b7(D13V), results in a higher degree of assembly of a multimeric

complex III and cytochrome b maturation, despite the relatively

lower abundance of the 11 kDa subunit. The higher levels of

cytochrome c, and ISP in the mutant containing Qcr7p-

~7(R101/G12V) as compared to YSM-qcr7~ are in agreement with the

hypothesis of an increase in the degree of assembly. The finding

that cytochrome c, is present a t relatively higher levels than the 11

kDa subunit when compared to the wild type may seem to contradict

the hypothesis of Hemrika e t al. (49) who suggest that the C-

terminus of the 11 kDa subunit anchors the cytochrome c,

subcomplex (45. 47, 65). However, the 11 kDa subunit does not

constitute the only contact site of the cytochrome c, subcomplex

with the bc, complex.

So how does the above model explain the findings of mutants

expressing Q c r 7 p ~ 1 4 and Qcr7p-AZO? These mutants display an

intermediate levei (approximately 60%) of 11 kDa protein. This fact

reinforces the notion stated earlier, that the contact site between

the Qcr7 protein and the 11 kDa subunit is beyond residue 8. One

may wonder why the interaction between Qcr7pA14, Qcr7p-A20 and

the 11 kDa subunit is stronger than that between the 11 kDa subunit

and Qcr7p-~7(D13V) or Qcr7p-~7(Rl OVG1 ZV), since Arg-1 O, Gly-12,

and Asp-13 are absent. Firstly, this could be taken as evidence for,

and could be explained by, the existence of another contact site

(beyond residue 15) between the Qcr7 protein and the 11 kDa subunit.

Secondly, the putative repulsive effect that is present between the

11 kDa subunit and the mutant proteins Qcr7p-A7(RlOI/G12V) and

Qcr7p-a7(D13V), de facto, is absent in Qcr7p-A14 and Qcr7p-~20.

The observation that mutants with Qcr7p-~14 and Qcr7pA20 have

significant levels of 11 kDa subunit, speaks for an association of the

Qcr7 protein and the 11 kDa subunit and against total misfolding of

Qcr7p-a14 and Qcr7pa20. The observation that the mutant with

Qcr7p-a20 has ISP and cytochrome c, levels comparable to YSM-

qcriA, whereas the level of 11 kDa subunit is significantly higher,

speaks for a direct interaction of the Qcr7 protein with the 11 kDa

subunit. The levels of ISP and cytochrome c, which are as low as in

YSM-qcr7~, furthermore constitute circumstantial evidence that

holocytochrome b is also undetectable in this mutant as it is in YSM-

Q C ~ ~ A (33). Hence, taken together al1 the evidence suggests that the

mutant containing Qcr7p-~20 is presumably the most compromised

of al1 the mutants that contain detectable Qcr7 protein in the

mitochondria. This argument is indirectly supported by the inability

of this mutant to grow on non-repressive carbon sources, such as

galactose.

The results of the current work suggest that the N-terminus of

the Qcr7 protein is involved in the assembly of complex III.

Although residues 1 to 7 (of the mature protein) seem tu play a role

in the assembly process, they are not crucial for the formation of a

functional bc,complex. In contrast, the region between residues 8

and 20 of the mature Qcr7 protein is essential for the functional

assembly of ubiquinol-cytochrome c oxidoreductase. In addition to

the role of the Qcr7 protein N-terminus in assembly, residues 2 to 8

(of the mature protein) may facilitate import into mitochondria or

confer stability to the protein. The elucidation of the function of

the subunit 7 N-terminus with respect to assembly may explain why

this region is retained as part of the mature protein rather than

being cleaved upon import into the mitochondria (if indeed it is

involved in import).

Future Directions

The current study indicates that the amino-terminus of

subunit 7 of the yeast bc, complex is essential for the formation of a

subcomplex consisting of cytochrome b, the 14 kDa and the 11 kDa

subunits. Without the formation of this subcomplex a functional

multimeric enzyme complex is not formed and mutant yeast display

a respiration-deficient phenotype. In addition to the proposed role

in assembly, the N-terminus of the Qcr7 protein is suggested to

increase the efficiency of the localization of subunit 7 into

mitochondria. Further studies can be perforrned t o confirm the

predicted functions of the Qcr7 protein amino-terminus.

Confirmation of assembly of the Qcr7 protein N-terminus with

cytochrome b and/or the 1 7 kDa subunit and identification of contact

sites

Confirmation of the putative interaction between cytochrome b

and the Qcr7 protein, as well as between the 11 kDa subunit and the

Qcr7 protein can be performed by CO-immunoprecipitation

procedures. The use of different conditions could not only confirm

the CO-precipitation of the components of the cytochrome b

subcomplex, but could potentially identify other subunits of complex

III that are in contact with either cytochrome b, the 14 kDa subunit,

or the 11 kDa subunit, respectively. For successful

immunoprecipitation it is important to obtain a good antibody.

Although obtaining a good antibody requires some luck, the

investigator should take into account the following information

when choosing the peptide that will be used to obtain the antibody.

The anti-Qcr7 protein antibody used in the immonoprecipitation

procedures of this study did not seem to CO-precipitate any other

subunits. This might be because the C-terminus (which contains the

epitope for the antibody) of the 14 kDa protein is buried in the

interior of complex III and/or because the C-terminus is believed to

anchor the core protein subcomplex (49. 66). Since the middle part of

the Qcr7 protein might be involved in forming a membrane-spanning

segment (481, raising an antibody against a reg ion immediately

following the Qcr7 protein N-terminus would be a plausible

possibility. Alternatively, it might be more useful t o raise an

antibody against cytochrome b or the N-terminus of the 11 kDa

subunit. The N-terminus of the 11 kDa subunit would probably be a

better choice than the C-terminus of the 11 kDa subunit, which has

been suggested to be located either on the matrix side of the IMM (5%

6 5 ) or on the IMS side of the IMM (66. 671, because the C-terminus has

been proposed to anchor the cytochrome c, subcomplex (49). Raising

an antibody against cytochrome b is difficult owing t o the

hydrophobicity of this protein and was unsuccessfully attempted

during this study. An antibody against cytochrome b would open up a

multitude of experimental possibilities (see below).

The residues involved in the interaction between the Qcr7

protein N-terminus and cytochrome b and/or the 11 kDa subunit

might be identified by the yeast "two-hybrid system". In this

experimental system, the Qcr7 protein amino-terminus can be used

to assay for the interaction with various regions of the 11 kDa

subunit and cytochrome b. The advantage of this system is the

inherent HA epitope tagging that can be used to detect fusion

proteins by Western blot analysis with commercially available

antibody. Hence, antibodies against the subcloned fragments are not

required. The disadvantage of the two-hybrid system is that it is

based on the solubility of proteins in aqueous environment. This

could be problematic for membrane proteins or for protein-protein

interactions that occur in a hydrophobic environment.

An anti-cytochrome b antibody can be used to further clarify

some of the results obtained in this study. Assaying for the levels

of apocytochrome b by Western blot analysis, versus holocytochrome

b by spectral analysis, could be used to gain further insight into the

mechanism of assembly. If results indicate that the levels of

apocytochrome b are low in strains expressing the N-terminally

truncated proteins of this study, then the respective truncated

regions of the Qcr7 protein could be implicated in the direct

assembly with cytochrome b. If, however the results showed that

apocytochrome b is present a t significantly higher steady-state

levels than holocytochrome b, then this would implicate the Qcr7

protein N-terminus in assembly a t the level of cytochrome b

maturation. In this case the Qcr7 protein N-terminus might be

involved in the stabilization of the cytochrome b heme environment

so as to maintain it in a state that is competent for the insertion of

heme/s.

Testing for the involvement of the Q u 7 protein N-terminus in

mitochondrial irnport

Based on the results of this study, the N-terminal seven amino

acids (after Met-1) o f the Qcr7 protein have been implicated in

mitochondrial import of this subunit. The truncation of seven or

more residues from the N-terminus resulted in decreased

mitochondrial steady-state levels o f the mutated Qcr7 proteins

from strains overexpressing the mutated genes as compared to the

strain overexpressing the wild type QCR7 gene. The functional

deficiencies observed in some of these strains expressing mutated

proteins cannot be attributed to the decreased levels of subunit 7 in

the mitochondria, as they are no lower than the levels observed in

the parental strain in which the QCR7 gene is not overexpressed.

Nevertheless, it is interesting t o investigate the cause for the

observed lowered steady-state levels.

To determine whether any of the lowered levels of the mutated

Qcr7 proteins are due t o a Iowered efficiency o f mitochondrial

import or to increased degradation, one could radioactively label

cytoplasmic proteins in vivo with [3SS]-methionine. After chasing

proteins for various times with cold medium, immunoprecipitation,

and SDS PAGE, one could distinguish whether the mutated Qcr7

proteins are accumulating in the cytoplasm and subsequently

degraded due to a decreased rate of import, or whether the mutant

proteins are imported at equal rates to the wild type, but degraded

faster inside the mitochondria due to an instability as a result of

mutation.

An alternative t o determining the efficiency of import in vivo,

is t o radiolabel proteins by in vitro translation in the presence of

reticulocyte lysate. Purified precursor protein can then be added to

mitochondria a t increasing concentrations in a standard import

mixture according to Millar and Shore (171). Following SDS PAGE,

import into mitochondria can then be tested for by resistance to

externally added proteases and the extent of import can be

determined by fluorography. To test whether the N-terminus of the

Qcr7 protein contains a mitochondrial targeting sequence, one could

fuse the N-terminal 21 amino acids of subunit 7 t o a non-

mitochondrial passenger protein such as the cytosolic mouse

dihydrofolate reductase (DHFR) (37). AS above, this import assay

could also be performed by in vitro synthesis of the chimeric Qcr7-

DHFR protein. Again, localization into mitochondria can then be

tested for by the resistance to externally added proteases.

An in vitro assay system can also be used to determine

whether the mutated Qcr7 proteins that are localized in the

mitochondria reach their final destination in the IMM or whether

they are accumulating in the matrix. Depending on whether

mitochondria or mitoplasts (mitochondria which lack the OMM) are

used in the in vitro assay in combination with protease resistance,

one can determine whether the Qcr7 proteins are associated with

the IMM or present in the matrix (142). According to Japa e t a l (142)

cytochrome b is required for the association o f the Qcr7 protein

with the membrane. Hence, if the mutated Qcr7 proteins are shown

to be localized in the matrix rather than in the membrane, then this

would further implicate the N-terminus in the direct assembly with

cytochrome b. If, however, the mutated Qcr7 proteins are localized

to the IMM, the obsewed assembly defect would more likely be a t the

level of cytochrome b maturation as discussed in the assembly

section above.

Further mutagenesis of the Qcr7 protein

Further mutational analysis of the Qcr7 protein in the context

of the full-length protein or in the context of a a7 truncation, can be

performed to confirm sorne of the predictions from this study. For

example, to identify which of the two residues, if not both, are

responsible for the p e t - phenotype in mutants with Qcr7p-

~7(R101/G12V) and Qcr7p-A7(RlOT/K44N), one would construct

mutants with full-length proteins Qcr7p-G1 ZV and Qcr7p-K44N.

Should these mutants display a wild type phenotype, then it would be

of interest to construct two mutants with the same residue

substitutions in the context of Qcr7p-~7. Should these mutants

compare t o the wild type as well, then the issue of whether

substitutions for Arg-1 O or both Arg-1 O and Gly-lZ/Lys-44

together are causing the p e t - phenotype could be resolved by

constructing mutants with Qcr7p-~7 containing substitutions for

Arg-10. In addition t o point substitutions a t the above residues, it

would also be of interest to construct a mutant with the full-length

Qcr7p-D'i3V. This will resolve the question as t o whether point

mutations are only informative in the context of Qcr7p-A7, or

whether they can disrupt assembly in the context of a full-length

protein. In addition, this will identify potentially important

residues.

If further analysis of the Qcr7 protein is desired, it might be

more informative in the future t o perform random mutagenesis of

the QCR7 gene rather than site-directed mutagenesis, since very few

of the mutants in this study displayed a respiration-deficient

phenotype. This can be performed by a number of methods, including

UV irradiation, PCR, and alanine scanning (1 57# 58). Alternatively,

further site-directed mutagenesis can be employed to mutate

consewed residues as determined by the comparison of homologues

of the Qcr7 protein (146). A preliminary screen for respiration-

deficient mutants can be performed by growth analysis on non-

fermentable carbon sources. Deficient mutants can then be further

characterized by the various methods outlined in the results

chapters and in the Future Directions of this dissertation. In order

to circumvent the issue of glucose repression which results in a

significant decrease of complex Ill-linked enzyme activities and in

the inability to perform spectra of the cytochromes (see Chapter 4),

mutant genes can be permanently integrated into the genome by

homologous recombination (as described in Chapter 2). This would

elirninate the need for selective growth in minimal media and the

mutant cells could be grown in rich medium containing non-

repressive carbon sources such as galactose. Further mutational

analysis of the Qcr7p-~7 amino-terminus (in which residues 2 t o 8

are deleted) has been performed by substituting residues 9 to 1 5

wi th a poly-alanine segment (So-Young Lee, personal

communication). In this experiment the Qcr7 protein is not

detectable in the rnitochondria and the assembly phenotype of the

mutant strain is comparable to that in YSM-qcr7a.

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