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Localization of the Tomato Bushy Stunt Virus ReplicationProtein p33 Reveals a Peroxisome-to-EndoplasmicReticulum Sorting Pathway W
AndrewW. McCartney,a John S. Greenwood,a Marc R. Fabian,b K. AndrewWhite,b and Robert T. Mullena,1
a Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canadab Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada
Tomato bushy stunt virus (TBSV), a positive-strand RNA virus, causes extensive inward vesiculations of the peroxisomal
boundary membrane and formation of peroxisomal multivesicular bodies (pMVBs). Although pMVBs are known to contain
protein components of the viral membrane-bound RNA replication complex, the mechanisms of protein targeting to
peroxisomal membranes and participation in pMVB biogenesis are not well understood. We show that the TBSV 33-kD
replication protein (p33), expressed on its own, targets initially from the cytosol to peroxisomes, causing their progressive
aggregation and eventually the formation of peroxisomal ghosts. These altered peroxisomes are distinct from pMVBs; they
lack internal vesicles and are surrounded by novel cytosolic vesicles that contain p33 and appear to be derived from
evaginations of the peroxisomal boundary membrane. Concomitant with these changes in peroxisomes, p33 and resident
peroxisomal membrane proteins are relocalized to the peroxisomal endoplasmic reticulum (pER) subdomain. This sorting of
p33 is disrupted by the coexpression of a dominant-negative mutant of ADP-ribosylation factor1, implicating coatomer in
vesicle formation at peroxisomes. Mutational analysis of p33 revealed that its intracellular sorting is also mediated by
several targeting signals, including three peroxisomal targeting elements that function cooperatively, plus a pER targeting
signal resembling an Arg-based motif responsible for vesicle-mediated retrieval of escaped ER membrane proteins from the
Golgi. These results provide insight into virus-induced intracellular rearrangements and reveal a peroxisome-to-pER sorting
pathway, raising new mechanistic questions regarding the biogenesis of peroxisomes in plants.
INTRODUCTION
Plus-sense, single-stranded RNA viruses infect most eukaryotes
and are the predominant class of viruses that infect plants.
Tombusviruses belong to a family of positive-strand RNA plant
viruses (Tombusviridae) that possess relatively small genomes
(;4800 nucleotides) and include Cymbidium ringspot virus
(CymRSV), Cucumber necrosis virus (CNV), Carnation Italian
ringspot virus, and Tomato bushy stunt virus (TBSV), the last of
which is probably the best studied in terms of its genome
replication and recombination (reviewed in White and Nagy,
2004). The TBSV genome contains five open reading frames
(ORFs) (Figure 1). ORF1 encodes a 33-kD auxiliary replication
protein (p33), and ORF2 encodes a 92-kD RNA-dependent RNA
polymerase (p92) and is produced by the translational read-
through of the p33 amber stop codon. Both p33 and p92 are
translated directly from the viral genome in infected cells and
interact asmembrane-bound components of theRNA replication
complex (K.B. Scholthof et al., 1995; Rajendran andNagy, 2004).
The remaining three ORFs in the TBSV genome encode a coat
protein of 41 kD (ORF3), a 22-kD protein required for cell-to-cell
movement of the virus (ORF4), and a 19-kD protein that functions
as a suppressor of virus-induced gene silencing (ORF5) (H.B.
Scholthof et al., 1995).
TBSV can infect a variety of plant species, and in all cases the
most conspicuous cytopathological feature of infected cells is
the presence of multivesicular bodies (MVBs) derived from
peroxisomes (reviewed in Martelli et al., 1988). These novel
intracellular structures (referred to herein as peroxisomal multi-
vesicular bodies [pMVBs]) form initially by a progressive inward
vesiculation of the boundary membrane of preexisting perox-
isomes, resulting in the organelle’s interior (matrix) housing up to
several hundred spherical to ovoid vesicles of 80 to 150 nm in
diameter. Eventually, the boundary membrane of individual
pMVBs also produces one or more large, spherical, and vesicle-
containing extrusions that fold back and engulf portions of the
cytosol, yielding doughnut-shaped or sometimes C-shaped
pMVBs that no longer resemble the peroxisomes from which
they are derived.
Although the progressive structural reorganization of perox-
isomes into pMVBs in TBSV-infected cells has been relatively
well documented, the functional role of these complex membra-
nous compartments and the molecular mechanism(s) under-
lying their biogenesis are largely speculative. For instance,
because pMVBs are frequently observed to be in close associ-
ation with segments of the endoplasmic reticulum (ER), this
1 To whom correspondence should be addressed. E-mail [email protected]; fax 519-837-2075.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Robert T. Mullen([email protected]).WOnline version contains Web-only data.Article, publication date, and citation information can be found atwww.plantcell.org/cgi/doi/10.1105/tpc.105.036350.
The Plant Cell, Vol. 17, 3513–3531, December 2005, www.plantcell.orgª 2005 American Society of Plant Biologists
endomembrane compartment has been proposed as the mem-
brane source for the numerous vesiculation events that occur at
the peroxisomal boundary membrane during TBSV infection
(Martelli et al., 1988). It has also been proposed that the small
vesicles that are formedwithin pMVBs are the sites of TBSVRNA
replication, because these structures can incorporate tritiated
uridine (Appiano et al., 1983, 1986) and could serve to protect
nascent viral transcripts from host cell RNases. Consistent with
this premise, both p33 and p92 are membrane-associated
proteins; thus, the TBSV replication complex is likely anchored
onto the internal vesicles of pMVBs (K.B. Scholthof et al., 1995).
However, how nascent p33 and p92 are initially targeted to
peroxisomal membranes and participate in pMVB formation
during the TBSV infection process has not been resolved. In fact,
the only significant insights to these events have come from
studies of homologs of p33 in the related CymRSV and CNV. For
instance, modified infectious clones of CymRSV were used to
demonstrate that the N-terminal portion (including the two
predicted membrane-spanning domains) of p33 is essential for
the formation of pMVBs (Burgyan et al., 1996; Rubino and Russo,
1998). The same N-terminal region of CymRSV and CNV p33s
was also shown to contain the targeting information essential for
their sorting to peroxisomal membranes in Saccharomyces
cerevisiae, although a specific targeting sequence in either
protein was not defined (Navarro et al., 2004; Panavas et al.,
2005).
We are interested in understanding all aspects of the TBSV life
cycle, including how this virus exploits peroxisomes as the sites
for viral RNA replication and what this might tell us about plant
peroxisome biogenesis in general. Previously, we and others
showed that newly synthesized peroxisomal membrane proteins
(PMPs) are sorted either directly to peroxisomes from the cytosol
or indirectly to peroxisomes by way of specialized regions of the
ER, known as peroxisomal endoplasmic reticulum (pER), and
pER-derived vesicles (reviewed in Trelease and Lingard, 2005).
Here, we provide evidence that the TBSV replication protein p33
makes use of a novel peroxisome-to-pER vesicle-mediated
sorting pathway. Using a combination of fluorescence and
electron microscopy, as well as site-directed mutagenesis
experiments, we show that the trafficking and molecular target-
ing signals of p33 are far more complex than previously sug-
gested, especially with respect to the involvement of pER.
Overall, these results using p33 as an investigative tool have
important implications for our overall understanding of pMVB
biogenesis and the biogenetic link between peroxisomes and
pER via vesicle transport in plants.
RESULTS
Peroxisomes in TBSV-Transformed BY-2 Cells Are Altered in
Terms of Their Distribution andMorphology, Contain Both
p33 and p92, and Serve as the Sites of Viral RNA Synthesis
To determine the subcellular localization of p33, we initially
examined the protein in plant cells transformedwith TBSV cDNA.
Toward that end, a plasmid containing the full-length cDNAof the
TBSV genome that can launch autonomous TBSV replication
(pHST20; Scholthof, 1999) was introduced by biolistic bombard-
ment into tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2)
suspension-cultured cells, serving as a model system for study-
ing protein sorting in vivo (Nagata et al., 1992). Expressed p33
along with its translation read-through product, p92, were
localized using fluor-conjugated secondary antibodies bound
to IgGs raised against a synthetic peptide that corresponds to
an amino acid sequence in both proteins (Figure 1, asterisk).
Antibody specificity for both p33 and p92 was confirmed by
immunoblotting of protein extracts from BY-2 protoplasts trans-
formed with TBSV RNA (Figure 2A).
Figure 2B is a grouping of representative micrographs illus-
trating that introduced p33 and p92 colocalized precisely with
the endogenous peroxisomal matrix protein catalase in individ-
ual TBSV-transformed BY-2 cells at 2, 4, 24, and 48 h after
bombardment. These time-course results also revealed dra-
matic and progressive alterations in peroxisome distribution and
morphology. For instance, at 2 and 4 h after bombardment, most
of the peroxisomes in TBSV-transformed cells were larger in size
and fewer in number compared with peroxisomes in neighboring
nontransformed cells. Close inspection revealed that these
globular-like peroxisomes consisted of several individual perox-
isomes (Figure 2B, inset), suggesting that they formed by co-
alescence of preexisting organelles.
Changes in peroxisome morphology in TBSV-transformed
cells were even more pronounced at 24 and 48 h after bom-
bardment; p33 and p92 colocalized with catalase in several large
globular and elongated peroxisomal structures that ranged in
size up to 30 mm in length, were typically restricted to the
perinuclear region of the cell, and had amore diffuse appearance
compared with the distinct aggregated peroxisomal structures
observed at earlier time points (Figure 2B). In contrast with
peroxisomes, the morphology and distribution of other sub-
cellular organelles in TBSV-transformed cells, including mito-
chondria, ER, plastids, and Golgi, were unaltered (Figure 2C).
To determine whether the novel peroxisomal structures in
TBSV-transformed BY-2 cells also contained double-stranded
RNA (dsRNA) intermediates produced during viral replication
(White and Nagy, 2004), cells were dual-labeled with anti-p33/
p92 IgGs and monoclonal antibodies raised against dsRNA.
Figure 2D shows that at 24 h after bombardment, dsRNA in
a TBSV-transformed cell localized exclusively to distinct portions
of the globular p33- and p92-containing peroxisomes, indicating
that viral RNA synthesis occurred at these sites, presumably
Figure 1. Diagram of the TBSV Genome.
The five ORFs coding for p33, p92, p41, p22, and p19 are represented as
boxes, and noncoding regions within the genome are represented as
lines. Read-through of the amber stop codon of p33 (stippled line) results
in the translation of the p92 fusion protein. p22 and p19 are encoded by
overlapping ORFs on the same subgenomic RNA molecule. The two
thick black lines near the 59 end of the genome represent the two putative
membrane-spanning domains in p33 and p92. The asterisk highlights the
relative position of the amino acid sequence used to generate antibodies
that recognize both p33 and p92.
3514 The Plant Cell
Figure 2. Localization of p33 and p92 Replication Proteins in TBSV-Transformed BY-2 Cells.
(A) Immunoblot analysis of TBSV-transformed BY-2 protoplasts. Purified protoplasts were either mock-transformed or transformed with full-length
TBSV RNA transcripts. After a 24-h incubation period, total protein was subjected to SDS-PAGE and immunoblotting with anti-p33/p92 IgGs. The
positions of p33 and p92, as well as the molecular masses (in kD) of marker proteins, are shown at right and left of the blots, respectively.
(B) Temporal dynamics of p33 and p92 localization in BY-2 cells. Cells transformed with TBSV cDNA were fixed in formaldehyde at 2, 4, 24, or 48 h after
biolistic bombardment and then processed for immunofluorescencemicroscopy. The yellow color in the merged images (bottom row) indicates obvious
colocalizations of expressed p33 and p92 (top row) and endogenous peroxisomal catalase (middle row) in the same cells. The inset shows an enlarged
portion of the micrograph identified with an asterisk; arrowheads in the inset denote several individual (punctate) peroxisomes that form part of a larger
globular structure. Bar ¼ 10 mm.
(C) Morphology and distribution of mitochondria, ER, plastids, and Golgi in TBSV-transformed BY-2 cells. Merged images of cells transiently
transformed with p33 and p92 ([a] to [d]; green fluorescence) at 24 h after bombardment and either (immuno)stained (red fluorescence) for endogenous
mitochondrial b-ATPase (a) or endogenous ER with concanavalin A conjugated to Alexa 594 (b) or cotransformed with either plastidial glutathione
reductase fused to the green fluorescent protein (GR-GFP; Chew et al., 2003) (c) or the Golgi nucleotide sugar transporter 1 fused to the yellow
fluorescent protein (GONST1-YFP; Baldwin et al., 2001) (d). Note the similarity in the b-ATPase and concanavalin A staining in TBSV-transformed and
neighboring nontransformed cells in (a) and (b). Note also that the fluorescence attributable to coexpressed GR-GFP (c) and GONST1-YFP (d) in the
merged images was pseudocolored red. Bar in (a) ¼ 10 mm.
(D) Colocalization of p33, p92, and dsRNA in BY-2 cells. The yellow color and arrowheads in the merged image (bottom) indicate obvious
colocalizations of expressed p33 and p92 with dsRNA in a representative TBSV-transformed cell at 24 h after bombardment. Bar ¼ 10 mm.
Trafficking of Tomato Bushy Stunt Virus p33 3515
pMVBs. Neither mock transformations nor omission of anti-p33/
92 and/or anti-dsRNA antibodies yielded immunofluorescence
(data not shown).
p33 Expressed Individually in BY-2 Cells Is Localized Initially
to Aggregated Peroxisomes and Then to a Subdomain of the
ERConcomitant with the Formation of Peroxisomal Ghosts
Similar to the results presented in Figure 2B, p33 expressed on
its own colocalized with endogenous catalase in individual and
aggregated (globular) peroxisomes at the earlier stages of
expression and sorting (i.e., 2 and 4 h) (Figure 3A), suggesting
that p33 targeted directly from its site of synthesis in the cytosol
to peroxisomes. By 24 h after bombardment, however, p33
localized to two distinct subcellular compartments: globular
peroxisomes that had continued to increase in size, and a re-
ticular network that was distributed throughout the entire cell
(Figure 3A). The lack of endogenous catalase in this p33-
containing reticulum suggests that it is not formed by a marked
distension or induced elongation/tubulation of globular perox-
isomes but instead is a separate subcellular compartment(s).
At 48 h after bombardment, p33 remained localized to a diffuse
reticular network as well as to several globular structures,
although the latter were generally smaller than those observed
at 24 h after bombardment and, thus, were more difficult to
discern because of the pervasive nature of the reticular fluores-
cence (Figure 3A). Interestingly, p33-transformed cells at 48 h
after bombardment also were devoid of endogenous catalase
(cf. catalase staining in neighboring nontransformed cells in
Figure 3A). Similar results were observed when these p33-
transformed cells were immunostained for the endogenous per-
oxisomal matrix enzyme isocitrate lyase (data not shown).
The eventual loss of peroxisomal matrix enzymes in cells
expressing p33 was unexpected and suggested that the perox-
isomes in these cells were degraded (e.g., pexophagy) (Farre and
Subramani, 2004). Alternatively, the integrity of the peroxisomal
boundary membranes in these cells may have been compro-
mised such that the small globular structures observed at 48 h
after bombardment were remnants of peroxisomes that pos-
sessed membrane proteins but lacked matrix proteins, similar to
the so-called peroxisomal ghosts described in yeast and mam-
malian cells having mutations in peroxisomal matrix protein
import (reviewed in Purdue and Lazarow, 2001). To test these
possibilities, p33-transfromed cells at 48 h after bombardment
were examined for the presence or absence of two well-
characterized PMPs, ascorbate peroxidase (APX) and a 22-kD
PMP (PMP22) (Lisenbee et al., 2003a; Murphy et al., 2003, and
references therein).
For dual-labeling experiments with p33 and APX, a myc
epitope tag was appended to the C terminus of p33 (p33-myc).
p33-myc resembles wild-type p33 in that it localized to both
a diffuse reticulum and several small globular structures within
the same cell at 48 h after bombardment (Figure 3B, a),
confirming that the addition of the myc epitope to p33 does not
disrupt its normal subcellular sorting. Endogenous APX was also
readily immunodetectable in a p33-myc–transformed cell, and
this resident PMP colocalized with p33-myc in the same small
globular structures (Figure 3B, a and b, closed arrowheads),
indicating that, although lacking endogenous matrix enzymes,
these structures contain APX and likely other PMPs (i.e., perox-
isomal ghosts). Interestingly, endogenous APX in the same
transformed cell also colocalized with p33-myc in the reticular
network (Figure 3B, c and d). By contrast, endogenous APX in
neighboring nontransformed cells localized at its steady state
only to normal individual peroxisomes, as expected (Mullen et al.,
1999) (Figure 3B, b).
Transiently expressed PMP22 was also immunodetectable in
p33-cotransformed cells, and similar to endogenous APX, the
subcellular localization of PMP22 in these cells was substantially
altered. That is, although N-terminal myc-tagged PMP22 (myc-
PMP22) expressed on its own localized exclusively to normal,
individual catalase-containing peroxisomes, as expected (Murphy
et al., 2003) (Figure 3B, e and f), coexpression of myc-PMP22 and
p33 resulted in both proteins localized to the same small globular
peroxisomal ghosts and reticular network (Figure 3B, g and h).
Given the distinct morphology of the reticular compartment to
which p33 (and endogenous APX and coexpressed PMP22 in
p33-transformed cells) localizes and the fact that at least some
plant PMPs, such as APX (Mullen et al., 1999; Nito et al., 2001;
Lisenbee et al., 2003a) and PEX16 (Karnik and Trelease, 2005),
sort indirectly to peroxisomes by way of pER, we speculated that
p33wasalso targeted toER, or a portion thereof. Figure 3C, a and
b, show that the reticular fluorescence patterns attributable to
expressed p33 and concanavalin A–stained ER did not coloc-
alize in the same cell at 48 h after bombardment. A lack of
colocalization was observed also between p33 and the endog-
enous ER resident protein calreticulin (data not shown). By
contrast, p33 colocalized entirely with coexpressed chloram-
phenicol acetyltransferase (CAT) fused to the 36 C-terminal
residues of APX (CAT-APX) (Figure 3C, c and d). This portion of
APX includes the targeting signals responsible for sorting this
PMP to peroxisomes via pER; thus, when CAT-APX is overex-
pressed, the fusion protein localizes throughout its entire sorting
pathway, including pER (Mullen et al., 1999, 2001; Mullen and
Trelease, 2000).
Sorting of p33 from Peroxisomes to pER Is Not Affected
by Cycloheximide or Cold Treatment but Is Disrupted by
a Dominant-Negative Mutant of ADP-Ribosylation Factor1
Although the data presented in Figure 3 are indicative of p33
sorting initially from the cytosol to peroxisomes and then to pER,
it is also possible that newly synthesized p33 sorted directly to
pER at later time points (e.g., 12 h after bombardment) by default,
as a result of the dramatic alterations in peroxisomal integrity
caused by p33 (over)expression and/or saturation of the host cell
machinery responsible for PMP (p33) targeting and integration.
To address this possibility, p33-transformed cells at 4 h after
bombardment were incubated with the protein synthesis in-
hibitor cycloheximide. We selected 4 h for these experiments
because p33 localizes specifically to peroxisomes at this time
(Figure 3A), and observations of p33 in pER in cells that were
subsequently incubated with cycloheximide would be convinc-
ing evidence that the protein sorts from peroxisomes to pER.
Figure 4A shows that expressed p33 at 12 h after bombard-
ment localized to both globular peroxisomes and pER in either
3516 The Plant Cell
Figure 3. Localization of p33 in BY-2 Cells.
(A) Temporal dynamics of p33 localization to peroxisomal structures and a reticular network in BY-2 cells. Transformed cells were fixed in formaldehyde
at 2, 4, 24, or 48 h after bombardment and then processed for immunofluorescence microscopy. The yellow color in the merged images indicates
obvious colocalizations of expressed p33 and endogenous catalase in the same cells. Arrowheads denote the locations of several small p33-containing
globular structures. Bar ¼ 10 mm.
(B) Localization of p33, APX, and PMP22 in BY-2 cells at 48 h after bombardment. Cells were transformed with either p33-myc ([a] and [c]) or myc-
PMP22 (e) or cotransformed with myc-PMP22 (g) and p33 (h). Transformed cells were also immunostained for either endogenous APX ([b] and [d]) or
endogenous catalase (f). The portions of the p33-myc–transformed cells outlined by the stippled box in (a) and (b) are shown at higher magnification in
(c) and (d). Closed arrowheads in (a) and (b), (c) and (d), (e) and (f), and (g) and (h) indicate obvious colocalizations. The open arrowhead in (b) denotes
endogenous APX localized exclusively in individual (punctate) peroxisomes in a nontransformed cell. Bar in (a) ¼ 10 mm.
(C) Localization of p33 to pER in BY-2 cells at 48 h after bombardment. Cells were either transformed with p33 ([a] and [c]) and costained for
endogenous ER with concanavalin A conjugated to Alexa 594 (b) or cotransformed with CAT-APX (d). Arrowheads in (c) and (d) indicate obvious
colocalizations. Bar in (a) ¼ 10 mm.
Trafficking of Tomato Bushy Stunt Virus p33 3517
Figure 4. Effect of Cycloheximide, a Dominant-Negative Mutant of Arf1, and Cold Shock on p33 Localization in BY-2 Cells.
(A) Localization of p33 and CAT-APX in cycloheximide-treated BY-2 cells. Cells were transformed with either p33 or CAT-APX and, at 4 h after
bombardment, either fixed in formaldehyde or incubated for an additional 8 h (12 h total) in the presence or absence of cycloheximide, followed
by formaldehyde fixation. Transformed cells were also immunostained for endogenous catalase. Arrowheads indicate obvious colocalizations. Bar ¼10 mm.
(B) A dominant-negative mutant of Arf1 (Arf1Q71L) blocks sorting of p33 and RFP-HDEL to ER (pER). BY-2 cells were cotransformed with p33 or RFP-
HDEL and wild-type Arf1 or Arf1Q71L. Transformed cells were fixed in formaldehyde at 24 h and stained with concanavalin A (ConA) conjugated to
Alexa 394. Closed arrowheads indicate obvious colocalizations. The differential interference contrast (DIC) image corresponds to the same cell
cotransformed with p33 and Arf1Q71L. Note that RFP-HDEL in cells coexpressing wild-type Arf1 localized entirely to concanavalin A–stained ER,
whereas RFP-HDEL in cells coexpressing Arf1Q71L localized to both ER and Golgi; open arrowheads denote individual Golgi complexes. Bar¼ 10 mm.
(C) Localization of p33 and BS14a in cold-treated BY-2 cells. Cells transformed with either p33 or GFP-BS14a were incubated at either 14 or 258C for 4 h
(p33) or 8 h (GFP-BS14a). Transformed cells were then fixed in formaldehyde and (immuno)stained for endogenous peroxisomal catalase or
endogenous ER with concanavalin A conjugated to Alexa 594. Closed arrowheads indicate obvious colocalizations. The differential interference
contrast image corresponds to the same cell transformed with GFP-BS14a at 258C. Note that GFP-BS14a localizes entirely to the Golgi at 258C, as
expected (Uemura et al., 2004), but localizes only to the ER at 148C. Bar ¼ 10 mm.
3518 The Plant Cell
the absence or presence of cycloheximide, indicating that the
peroxisome is an intermediate sorting site for p33. In a positive
control experiment, CAT-APX expressed in the absence of
cycloheximide for 12 h localized throughout its sorting pathway,
including (aggregated) peroxisomes and pER, as expected
(Mullen et al., 1999) (Figure 4A). In the presence of cyclohexi-
mide, however, CAT-APX accumulated only in peroxisomal
aggregates, consistent with the sorting of this PMP to per-
oxisomes via pER. Additional control experiments with the
Golgi-localized SNARE, BS14a (green fluorescent protein
[GFP]-BS14a), confirmed the effect of cycloheximide on protein
synthesis and protein sorting in transformed BY-2 cells (see
Supplemental Figure 1 online).
The sorting of p33 fromperoxisomes to pERwas also analyzed
using a dominant-negative mutant of the ADP-ribosylation fac-
tor1 (Arf1Q71L) that inhibits multiple steps in the secretory
protein transport pathway by disrupting the formation of coat
protein complex I (COPI) coated vesicles (Pepperkok et al., 2000;
Takeuchi et al., 2002). Specifically, we tested whether blocking
COPI vesicle transport with Arf1Q71L changed the steady state
localization of coexpressed p33. Figure 4B shows that at 24 h
after bombardment, p33 coexpressedwith wild-type Arf1 did not
alter the viral protein’s normal localization to (globular) perox-
isomes and pER (cf. with the localization of p33 expressed on its
own at 24 h after bombardment in Figures 3A and 3B). By
contrast, coexpression of p33 with Arf1Q71L altered p33’s
localization at 24 h after bombardment from peroxisomes and
pER to entirely peroxisomes (Figure 4B), indicating that Arf1 is
necessary for sorting p33 from peroxisomes to pER. Similarly,
localization of the ER marker protein RFP-HDEL was altered
when coexpressed with Arf1Q71L (Figure 4B), consistent with
COPI being involved in the retrieval of escaped reticuloplasmins
from the Golgi back to the ER (Denecke, 2003).
To test whether p33 sorts initially to pER and then to perox-
isomes, but does so in such a transient manner that, unlike CAT-
APX, it is not readily detectable in pER, we examined the
localization of p33 at early time points after bombardment (i.e.,
4 h) in transformed cells incubated at 148C. Cold treatment,
among other cellular effects, disrupts protein transport from the
ER by inhibiting ER-derived vesicle formation (Bar-Peled and
Raikhel, 1997; Boevink et al., 1999; Phillipson et al., 2001). Thus,
if p33 sorts to peroxisomes by way of the ER (pER), then cold
treatment of p33-transformed cells should adversely affect this
sorting step and p33 should, at least partially, accumulate in
pER. However, as shown in Figure 4C, the sorting of p33 was
identical when cells were maintained 4 h after bombardment at
either 14 or 258C (i.e., p33 localized exclusively to peroxisomes at
both temperatures). Similar results were observed when p33-
transformed cells were incubated for 4 h after bombardment at
88C (data not shown). By contrast, sorting of GFP-BS14a from
ER to Golgi was disrupted in cells incubated at 148C (Figure 4C).
p33 Expressed in Tobacco Leaves Is Localized to the
Boundary Membranes of Aggregated Peroxisomes and
Novel Cytosolic Vesicles
As noted in the Introduction, TBSV causes progressive rear-
rangements of peroxisomeboundarymembranes that eventually
give rise to the formation of pMVBs (Martelli et al., 1988). Figures
5A to 5C illustrates the results of a series of replicate experiments
that served as a comparative control for this study. Nicotiana
benthamiana plants were rub-inoculated with TBSV RNA, and
systemically infected leaves were analyzed by transmission
electron microscopy. The representative sectional view of the
individual mesophyll cell in Figure 5A shows a cluster of five large
(3 to 5 mm) doughnut-shaped pMVBs, each consisting of
an extended membranous appendage encircling or engulfing a
large portion of the cytosol. Numerous 40- to 170-nm-diameter
vesicles are present within the lumen of each of these pMVBs,
either separate from or, on occasion, connected to the boundary
membrane (Figure 5B, inset), suggesting that they formed by
membrane invagination and vesiculation. Occasionally, pMVBs
were also found in close association with tubular membranous
structures that resembled the ER (Figure 5C), consistent with the
presumption that a biogenetic link exists between these two
compartments (Navarro et al., 2004). All other subcellular organ-
elles were unaltered in TBSV-infected cells. Controls, including
mock-rub-inoculated plants and wild-type untreated plants, did
not yield any noticeable changes in peroxisomal ultrastructure
(see the wild-type peroxisomes shown in Figures 5K and 5L).
Based on the immunofluorescence results shown in Figure 3A,
the large globular peroxisomal structures in p33-transformed
BY-2 cells at 24 h after bombardment are most likely pMVBs;
thus, p33 appears to be sufficient in forming pMVBs in the
absence of other TBSV proteins. However, testing this hypoth-
esis directly at the ultrastructural level using transiently trans-
formed BY-2 cells was difficult because of the low frequency of
cell transformation (0.5%) achieved by biolistic bombardment.
To circumvent this problem, p33 was expressed transiently in
tobacco leaves via Agrobacterium tumefaciens infiltration, a
method that yields transformation frequencies of nearly 100%
(Wroblewski et al., 2005). In addition, two GFP fusion proteins
were used, GFP-SKL (GFP linked to the C-terminal tripeptide
SKL, a prototypic matrix peroxisomal targeting signal) and p33-
GFP (full-length p33 linked to the N terminus of GFP), as vital
markers that provided a convenientmeans of assessing changes
in peroxisome morphology and distribution in infiltrated leaf
tissue before processing for electron microscopy. Three days
after Agrobacterium infiltration, GFP-SKL exhibited a punctate
fluorescence pattern consistent with this protein being localized
to matrices of individual peroxisomes (Figure 5D). By contrast,
p33-GFP exhibited a torus and globular fluorescence pattern
indicative of localization to the boundary membranes of a cluster
of aggregated peroxisomes (Figure 5F, inset), similar to the
localization of wild-type p33 to aggregated peroxisomes in BY-2
cells at 4 h after bombardment (Figure 3A).
Consistent with these fluorescence microscopy results, elec-
tron microscopic analysis of mesophyll cells transformed with
p33-GFP revealed that peroxisomes were highly aggregated,
consisting of clusters of 3 to 8, and up to 15, individual organelles
that varied in size from 200 to 1500 nm in diameter (Figures 5H to
5J). The interiors of these aggregated peroxisomes were as
devoid of vesicles as those observed in pMVBs (cf. Figures 5B
and 5H) and were only slightly more structured and electron
dense than the matrices of wild-type peroxisomes in mesophyll
cells either infiltrated with Agrobacterium containing empty
Trafficking of Tomato Bushy Stunt Virus p33 3519
Figure 5. Electron and Fluorescence Microscopic Analysis of Tobacco Mesophyll Cells Transformed with TBSV or p33-GFP.
(A) to (C) Electron micrographs of N. benthamiana leaves systemically infected with TBSV RNA. Arrowheads in (A) denote individual pMVBs in a TBSV-
infected mesophyll cell. The asterisk in (B) indicates a portion of the pMVB shown at higher magnification in the inset: arrowheads denote two distinct
vesicle-like structures located within the lumen of the pMVB that appear to be connected by a neck to the pMVB boundary membrane. Arrowheads in
(C) highlight a portion of tube-like ER adjacent to two pMVBs.
(D) to (G) Tobacco leaves infiltrated with Agrobacterium containing either GFP-SKL ([D] and [E]) or p33-GFP ([F] and [G]). The inset in (F) shows at
higher magnification the torus fluorescence pattern attributable to p33-GFP in the boundary membranes of several aggregated peroxisomes; compare
with the solid punctate fluorescence attributable to GFP-SKL in the matrix of several individual peroxisomes in (D). Differential interference contrast
images shown in (E) and (G) correspond to the same cells shown in (D) and (F), respectively.
(H) to (L) Tobacco leaves infiltrated with Agrobacterium containing either p33-GFP ([H] to [J]) or empty binary vector (pMAT037) (K) or nontransformed
(L). Asterisks in (H) to (J) denote accumulations of cytosolic vesicles adjacent to aggregated peroxisomes in p33-GFP–transformed mesophyll cells.
Arrowheads in (H) and (J) indicate examples of evaginations in the boundary membranes of aggregated peroxisomes. C, chloroplast; CW, cell wall; Cyt,
cytosol; G, Golgi; M, mitochondrion; P, peroxisome; V, vacuole. Bars in (A) to (C) and (H) to (L) ¼ 0.5 mm; bar in (D) ¼ 10 mm.
binary vector (Figure 5K) or nontransformed (Figure 5L). On the
other hand, the boundary membranes of aggregated peroxi-
somes in p33-GFP–transformed cells were largely distorted, with
contours that gave a rippled or scalloped complexion and
numerous evaginations (Figures 5H and 5J, arrowheads), sug-
gesting that nascent vesicles are externalized into the cytosol.
Consistent with this finding, collections of small (40 to 150 nm),
single membrane-bound vesicles were located in the cytosol
immediately adjacent to the aggregated peroxisomes (Figures
5H to 5J, asterisks). Similar cytosolic vesicles were detected in
mesophyll cells transformed with wild-type p33 (data not shown)
but were not observed in nontransformed cells (Figure 5L), cells
transformedwith vector alone (Figure 5K), or TBSV-infected cells
containing pMVBs (Figures 5A to 5C). Thus, although p33 is
responsible for the formation of the novel cytosolic vesicles, they
are not topologically equivalent to those formed during the
biogenesis of a so-called classical pMVB.
Immunogold labeling of the p33-GFP–transformed samples
represented in Figure 5 was performed to determine the precise
subcellular localization of the viral fusion protein. Virtually all of
the gold particles decorated the periphery (membrane) of the
novel cytosolic vesicles and their neighboring aggregated per-
oxisomes, aswell as thematrix of these peroxisomes (Figure 6A).
All other subcellular structures in these p33-GFP–transformed
cells in which the total surface area was several orders of
magnitude larger than that of the aggregated peroxisomes and
their surrounding vesicles displayed significantly lower frequen-
cies of gold particle labeling (Figures 6B and 6C), equivalent to
that observed when samples were incubated with preimmune
serum (Figure 6D).
The N-Terminal Half of p33 Possesses Both Peroxisome
and pER Targeting Information
To define the targeting information responsible for sorting p33 to
various subcellular compartments (i.e., peroxisomes and pER),
we conducted a series of targeting experiments using chimeras
consisting of different portions of p33 fused to CAT serving as
a passenger protein (Figures 7A and 7B). Although CAT alone
localized only to the cytosol of a transformedBY-2 cell, p33-CAT,
consisting of full-length p33 fused to the N terminus of CAT,
localized exclusively to aggregated (globular) peroxisomes.
These data for p33-CAT are consistent with the peroxisomal
localization of wild-type p33 in BY-2 cells at the same time point
(i.e., 4 h after bombardment; Figure 3A), indicating that the CAT
moiety does not affect p33 sorting. Similar to p33-CAT, p33
1–156-CAT, consisting of residues 1 to 156 of the p33
N terminus, including the N-terminal hydrophilic domain, trans-
membrane domains (TMDs) 1 and 2, and their intervening
hydrophilic loop sequence appended to CAT, localized to
globular peroxisomes. CAT-p33 131–296, however, was cyto-
solic (data not shown), suggesting that, unlike the N-terminal half
of p33, the C-terminal half of the protein lacks sufficient targeting
information.
p33-CAT fusion constructs consisting of smaller portions of
the p33 N-terminal 1 to 156 amino acid domain also targeted to
Figure 6. Localization of p33-GFP in Tobacco Mesophyll Cells.
Electron immunogold microscopy of tobacco leaves infiltrated with Agrobacterium containing p33-GFP. Thin sections represented in Figure 5 were
incubated with anti-p33/p92 IgGs ([A] to [C]) or preimmune serum (D) and protein A conjugated to 5-nm gold particles. The arrowheads in (A) denote
gold particles localized selectively on either cytosolic vesicles or aggregated peroxisomes, whereas the arrowheads in (B) to (D) denote gold particles
located infrequently on these or other subcellular regions (e.g., chloroplasts andmitochondria). The asterisk in (D) denotes the accumulation of cytosolic
vesicles adjacent to aggregated peroxisomes in a p33-GFP–transformed mesophyll cell. C, chloroplast; CW, cell wall; Cyt, cytosol; M, mitochondrion;
P, peroxisomes. Bars ¼ 0.5 mm.
Trafficking of Tomato Bushy Stunt Virus p33 3521
peroxisomes, albeit in an inefficientmanner (i.e., both p33 1–103-
CAT and p33 104–156-CAT localized to the cytosol and only
partially colocalized with endogenous catalase in globular and
individual peroxisomes) (Figure 7). By contrast, p33 1–75-CAT,
which includesmost of the 82–amino acid N-terminal hydrophilic
domain of p33, did not target to peroxisomes but instead
targeted entirely to pER, as indicated by its colocalization with
coexpressed GFP-APX, a fusion protein that, like CAT-APX
(Figure 3B), includes the 36 C-terminal amino acids of APX and
serves as a well-defined marker for pER while en route to
peroxisomes (Lisenbee et al., 2003b). Smaller portions of the
N-terminal 75 amino acid residues of p33 were not sufficient in
sorting CAT to pER or to peroxisomes (data not shown).
To define precisely the regions within the N-terminal half
(residues 1 to 156) of p33 responsible for its targeting to
peroxisomes and pER, we examined amino acid sequences
within the protein that resemble a prototypic membrane perox-
isomal targeting signal (mPTS) present in most other PMPs (i.e.,
a stretch of several positively charged residues adjacent to at
least one TMD) (reviewed in Trelease, 2002). We also focused on
sequences of the p33 homologs from CymRSV and CNV that
include the protein’s two TMDs and adjacent hydrophilic do-
mains and that were necessary for targeting to peroxisomes in
yeast cells (Navarro et al., 2004; Panavas et al., 2005). Alignment
of the deduced amino acid sequences for p33 from TBSV,
CymRSV, and CNV (Figure 8A) revealed a high degree of identity,
including two positively charged domains (residues 76 to 80 and
124 to 130; shaded in gray in Figure 8A) that resembled a pro-
totypic mPTS and that were located within the sequences
implicated in the sorting of CymRSV and CNV p33s to perox-
isomes.
To test whether these two positively charged domains in TBSV
p33 conferred necessary peroxisomal targeting information, all
of the basic residues at each region (underlined, -KRRQR- and
-RPSVPKK-) or at both regions were replaced with noncharged
Gly residues, and the efficiency with which the resulting mutant
proteins sorted to peroxisomes at 4 h after bombardment was
compared with that of wild-type p33 (Figures 8B and 8C). Unlike
wild-type p33, which localized only to (globular) peroxisomes at
4 h after bombardment (Figure 3A), p33-K76R77R78R80DG, p33-
R124K129K130DG, and p33-K76R77R78R80R124K129K130DG local-
ized to both globular peroxisomes (Figure 8C, a to f) and pER, as
indicated by their partial colocalizations with endogenous cata-
lase (Figure 8C, a to f), and coexpressed CAT-APX (data shown
for p33-K76R77R78R80R124K129K130DG only; Figure 8C, g and h).
These results indicate that although -K76R77R78R80- and
Figure 7. Localization of p33-CAT Fusion Proteins in BY-2 Cells.
(A) Scheme of various p33-CAT fusion proteins and their corresponding subcellular localization at 4 h after bombardment as peroxisomes (Perox),
cytosol (Cyt), or pER. The numbers in the name of each fusion construct denote the specific amino acid residues from p33 that were fused to the N or C
terminus of CAT. Black boxes in the illustrations denote the two putative TMDs in p33, with the numbers shown above p33-CAT indicating their relative
amino acid positions. Gray boxes denote bacterial CAT.
(B) Representative fluorescence patterns attributable to various constructs illustrated in (A). Each micrograph is labeled at top left with the name of the
transiently expressed (truncated) p33-CAT fusion construct, the endogenous peroxisomal marker protein, catalase, or coexpressed GFP-APX as
a marker protein for pER. The portions of the cells outlined by the stippled boxes in the bottom left two panels are shown at higher magnification in the
bottom right two panels. Data are shown for only selected constructs. Arrowheads indicate obvious colocalizations. Bar ¼ 10 mm.
3522 The Plant Cell
Figure 8. Localization of Modified Versions of p33 in BY-2 Cells.
(A) Amino acid sequence alignment of TBSV, CymRSV, and CNV p33s. Deduced amino acid sequences were obtained from GenBank and aligned using the
ClustalWalgorithm. Identical aminoacid residues ineachprotein are indicatedbyasterisks, and similar residuesare indicatedbycolons. TMDs (underlined; amino
acidresidues83to98and132to154)were identifiedusingTMHMM(version2.0)andvisual inspection.Aminoacidsequences inTBSVp33(andthecorresponding
residues in CymRSV and CNV p33s) proposed to be part of essential targeting signal elements and substituted with Gly residues are shaded gray or boxed.
(B) Subcellular localization of p33 mutants in BY-2 cells at 4 h after bombardment as peroxisomes (Perox), cytosol (Cyt), or pER. The numbers in the
name of each p33 mutant denote the specific amino acid residues that were replaced with Gly residues.
(C) Representative fluorescence patterns attributable to various constructs illustrated in (B). Cells transformed with a p33 mutant ([a], [c], [e], [i], [k],
[m], and [q]) or cotransformed with a p33 mutant ([g], [o], and [s]) and CAT-APX (serving as a pER marker protein) ([h], [p], and [t]) were fixed in
formaldehyde at 4 h after bombardment and then processed for immunofluorescence microscopy. Data are shown for only selected constructs.
(a) to (f) Transiently expressed p33-K76R77R78R80DG (a), p33-R124K129K130DG (c), p33-K76R77R78R80R124K129K130DG (e), and endogenous catalase
([b], [d], and [f]).
Trafficking of Tomato Bushy Stunt Virus p33 3523
-R124K129K130- in p33 are essential for its efficient sorting to
peroxisomes, additional targeting information exists within the
protein.
Because the N-terminal half of p33 is sufficient for sorting CAT
to peroxisomes (p33 1–156-CAT; Figure 7), any peroxisomal
targeting information in p33, in addition to regions 76 to 80 and
124 to 130, is likely located within this portion of the protein.
Indeed, a further inspection of the p33 N terminus revealed two
dibasic motifs (-K5R6- and -K11K12-; boxed in Figure 8A) that
resembled nonprototypic mPTSs identified previously in the
N termini of Arabidopsis thaliana and mammalian PMP22s
(Brosius et al., 2002; Murphy et al., 2003). These PMP22 mPTSs
are considered unique from the more common basic cluster
mPTS because they consist of a smaller stretch of positively
charged residues and are not immediately adjacent to a TMD.
Substitutions at both of these dibasic motifs in p33 with Gly
residues resulted in the mutant protein (p33-K5R6K11K12DG)
being only partially colocalized with endogenous peroxisomal
catalase (Figure 8C, i and j), indicating that this region is essen-
tial for efficiently sorting p33 to peroxisomes. Notably, p33-
K5R6K11K12DG, unlike the other p33 mutants described above,
also partially localized to the cytosol, rather than to pER (i.e., p33-
K5R6K11K12DG did not colocalize with coexpressed CAT-APX in
pER) (see Supplemental Figure 2 online), suggesting that this
region also contains (overlapping) targeting information neces-
sary for sorting p33 to pER.
The partial targeting of p33-K5R6K11K12DG to peroxisomes
was not unexpected, because at least two other peroxisomal
targeting elements within the protein (i.e., -KRRQR- and
-RPSVPKK-) were left intact. Partial localization of p33 to per-
oxisomes was also observed when either of these two regions
was mutated in the context of p33-K5R6K11K12DG (i.e., p33-
K5R6K11K12K76R77R78R80DG and p33-K5R6K11K12R124K129K130
DG both localized to peroxisomes and the cytosol; data not
shown). Only when all three of the regions essential for efficient
peroxisomal targeting of p33 were altered was the resulting
mutant protein (p33-K5R6K11K12K76R77R78R80R124K129K130DG)
no longer targeted to peroxisomes but instead accumulated
entirely in the cytosol (Figure 8B, k and l). Similar results were
observed when the same mutations were introduced into p92
(data not shown), indicating that both protein components of the
TBSV replication complex were targeted to peroxisomes in an
identical manner.
We further investigated the putative pER targeting signal within
the -K5R6K11K12- region of p33 by replacing each pair of basic
residues with Gly residues (underlined, -KRMIWPKK-). Similar to
p33-K5R6K11K12DG, p33-K11K12DG localized to peroxisomes
and pER (Figure 8C, m to p), but p33-K5R6DG localized to
peroxisomes and cytosol (Figure 8C, q to t). Similar substitutions
of -K5R6- with Gly residues in the context of the pER-localized
fusion protein p33 1–75-CAT (Figure 7) also resulted in the
mutant (p33 1–75K5R6DG-CAT) beingmislocalized entirely to the
cytosol (data not shown). Together, these data are consistent
with the -K5R6- region of p33 being essential for sorting to pER.
Disruption of p33 Targeting Signals Negatively Affects the
Replication of TBSV-Defective Interfering RNAs
The identification of distinct targeting sequences within p33
prompted us to investigate whether or not p33’s function in the
replication of viral RNAs was impaired when it was mislocalized
within the cell. Toward this end, in vivo trans-complementation
assays (Oster et al., 1998; Fabian and White, 2004) were used to
quantify viral RNA accumulation after infection under conditions
in which either p33 only was mutated or both p33 and p92 were
mutated.
Figure 9A shows the results of an analysis of viral RNA
replication with a wild-type p92 and different mutant versions
of p33 containing Gly substitutions in either one or combinations
of two or all three of the regions necessary for efficient perox-
isomal and/or pER targeting (Figure 8) (i.e., -K5R6K11K12-,
-K76R77R78R80-, and -R124K129K130-). Viral RNA replication was
assessed by detection and quantification of the accumulation of
a small viral RNA replicon, DI-72, which requires both p33 and
p92 for its replication (White and Morris, 1994). Compared with
cells expressing wild-type p33, those producing mutated forms
of p33 with modifications in one or more of the three targeting
regions showedprogressively reducedDI-72RNA levels (Figures
9A and 9C). Indeed, virtually no DI-72 RNA was detected in cells
expressing the triple mutant p33-K5R6K11K12K76R77R78R80R124
K129K130DG.
In Figure 9B, the replication of DI-72 was investigated under
conditions in which both p33 and p92 contained the targeting
mutations described above. With the exception of mutant p33/
p92-K76R77R78R80DG, the p33/p92 mutants showed similar or
more pronounced decreases in DI-72 RNA accumulation levels
than were observed for the p33 mutants (Figure 9C). Moreover,
DI-72 RNAs accumulated least in all double mutants, the triple
mutant, and the corresponding single p33 mutant that pos-
sessed alterationswithin the region responsible for pER targeting
(i.e., -K5R6K11K12-). These results indicate that robust viral RNA
replication requires proper peroxisome and/or pER targeting,
Figure 8. (continued).
(g) and (h) Transiently coexpressed p33-K76R77R78R80R124K129K130DG (g) and CAT-APX (h).
(i) to (n) Transiently expressed p33-K5R6K11K12DG (i), p33-K5R6K11K12K76R77R78R80R124K129K130DG (k), p33-K11K12DG (m), and endogenous catalase
([j], [l], and [n]).
(o) and (p) Transiently coexpressed p33-K11K12DG (o) and CAT-APX (p).
(q) and (r) Transiently expressed p33-K5R6DG (q) and endogenous catalase (r).
(s) and (t) Transiently coexpressed p33-K5R6DG (s) and CAT-APX (t).
Arrowheads indicate obvious colocalizations. Bar in (a) ¼ 10 mm.
3524 The Plant Cell
and they are consistent with the concept that such targeting
contributes to the proper assembly of the viral RNA replication
complex.
DISCUSSION
For viruses to successfully replicate within host cells, they must
manipulate and coordinate a variety of complex cellular path-
ways. As a result, viral infection often leads to an enhancement of
eventswithin the cell that are otherwise intractable to study; thus,
viruses and individual viral proteins can serve as unique tools to
investigate these cellular processes. Therefore, we used TBSV
as a means to investigate peroxisome biogenesis, a little un-
derstood event, specifically by focusing on the replication pro-
tein p33, which was demonstrated previously to be essential for
pMVB formation (Burgyan et al., 1996; Rubino and Russo, 1998).
Our findings support the notion that p33 successfully appropri-
ates different peroxisomal protein sorting pathways to facilitate
viral replication, including a previously unknown peroxisome-
to-pER sorting pathway.
p33 Causes Vesiculation of the Peroxisomal
Boundary Membrane
TBSV p33 expressed on its own caused the pronounced
accumulation of novel peroxisome-associated cytosolic vesicles
(Figure 5). These vesicles contained p33 (Figure 6), were dis-
persed among populations of aggregated peroxisomes, and
Figure 9. RNA Gel Blot Analysis of Progeny Viral RNAs Isolated from
Cucumber Protoplasts Inoculated with Various Mutant p33 RNA Tran-
scripts.
(A) Analysis of trans-expression activities of p33 mutants. Cucumber
protoplasts were cotransfected (via electroporation) with the appropriate
transcripts, and total nucleic acids were isolated after a 24-h incubation
and subjected to gel electrophoresis, RNA gel blotting, and autoradiog-
raphy. RNA transcripts used in the inoculations included the full-length
TBSV genome; DI83DII encoding wild-type p33; DI83DIIN, which is
identical to DI83DII except that a NcoI site was added to the 59 end of the
p33 ORF to allow for convenient cloning of the p33 mutants; DI83DIIN-
p33 mutants with Gly substitutions in either one or a combination of two
or all three of the regions necessary for targeting (the numbers in the
name of each mutant denote the specific amino acid residues that were
replaced with Gly residues); HS175, encoding wild-type p92; and DI-72.
Note that together, DI83DIIN (or DI83DII) and HS175 encode a functional
TBSV replicase capable of amplifying DI-72 to detectable levels. The
relative positions of TBSV genomic RNA (TBSV genome) and the two
TBSV subgenomic RNAs (sg1 and sg2 RNA) are shown at left, and the
position of DI-72 RNA is indicated at right.
(B) Analysis of trans-expression activities of p33/p92 mutants in the
context of the full-length TBSV genome. Cucumber protoplasts were
cotransfected with DI-72 and either RTD23, RTD23N, or RTD23N-p33/
p92, and DI-72 RNAs were analyzed by RNA gel blotting/autoradiogra-
phy as described for (A). RTD23 and RTD23N maintain the coding
sequence for p33 and the read-through sequence of p92, which allows
for p33 targeting sequence mutations to be integrated into both ORFs
simultaneously. Similar to DI83DIIN in (A), RTD23N contains a NcoI site
at the 59 end of the p33/p92 ORF for convenient cloning of p33/p92
mutants. The relative positions of RTD23 and DI-72 RNAs are shown at
left.
(C) Relative accumulation levels of DI-72 produced by the TBSV
replicase in the context of wild-type or mutant versions of either p33
(DI83DIIN) in (A) (black bars) or p33 and p92 (RTD23N) in (B) (white bars).
DI-72 accumulation levels in cells transfected with mutants are shown
relative to the accumulation levels obtained from wild-type p33 (DI83-
DIIN) or p33 and p92 (RTD23N) and were obtained using a phosphor
imager.
Trafficking of Tomato Bushy Stunt Virus p33 3525
most likely arose from the peroxisomal boundary membrane, as
also suggested for the novel vesicles observed in plant cells
transformed with CymRSV p33 (Bleve-Zacheo et al., 1997).
Because peroxisomes normally undergo outward vesiculation
or budding, albeit at a low frequency (Jedd and Chua, 2002), it
appears that p33 usurps the host cell molecular machinery
needed to accomplish peroxisome budding. However, the
identity of these host cell components and how they contribute
to peroxisome vesiculation remain to be determined.
The outward vesiculation of the peroxisomal boundary mem-
brane causedbyp33expressedon its ownprovides a reasonable
explanation for the relocalization of this viral protein, and of host
cell PMPs, from peroxisomes to pER (Figure 3B). The redistri-
bution of APX andPMP22 (alongwith p33) from their steady state
localization in peroxisomes (Mullen et al., 1999; Murphy et al.,
2003) to pER is likely attributable to their incorporation into the
peroxisome-derived vesicles that, after release into the cytosol,
traffic to and fuse with pER. The outward vesiculation of
peroxisomal membranes may also explain the formation of
peroxisomal membrane remnants or ghosts in p33-transformed
cells (Figure 3B); vesiculation may compromise the organelle’s
integrity such that the endogenous matrix constituents (e.g.,
catalase and isocitrate lyase) are either degraded and/or diluted
in the cytosol to below immunodetection. Indeed, transient
expression of a peroxisomal matrix marker protein (RFP-SKL)
in p33-cotransformed BY-2 cells resulted in the marker protein
being completely mislocalized to the cytosol at later time points
(e.g., 48 h; data not shown), suggesting that peroxisomal matrix
constituents, unlike p33 and host cell PMPs, are not incorpo-
rated into peroxisome-derived vesicles destined for pER.
p33 Uses a Peroxisome-to-pER Sorting Pathway in Plants
All plant PMPs examined seem to be synthesized in the cytosol
on free polyribosomes and sorted either directly to peroxisomes
or indirectly to peroxisomes via pER and pER-derived vesicles
(reviewed in Trelease and Lingard, 2005). One of the most
remarkable findings of this study is that p33 sorted initially from
the cytosol to peroxisomes and then to pER, presumably via
peroxisome-derived vesicles. Although one could argue that the
peroxisome-to-pER sorting of p33 was the result of protein
overexpression, results from several sources suggest otherwise.
For instance, in contrast with APX,which at early time points after
biolistic bombardment of BY-2 cells is readily detectable in pER
while en route to peroxisomes (Mullen et al., 1999) (Figure 4), p33
was not detectable in pER at similar times, even when cells were
cold-treated to block ER (pER) protein transport (Figures 3A and
4C). p33 also did not insert into purified microsomal membranes
in vitro (data not shown), whereas APX does so in an efficient
manner (Mullen et al., 1999), indicating that pER is not an
authentic sorting site for newly synthesized p33 in the cytosol.
Furthermore, experiments with cycloheximide (Figure 4A) and
a dominant-negative mutant of Arf1 (Arf1Q71L) (Figure 4B)
yielded the most convincing evidence that p33 localized in
peroxisomes is eventually sorted to pER.
Although our conclusion for peroxisome-to-pER sorting of
TBSV p33 is different from that drawn by Navarro et al. (2004) for
the sorting of CymRSV p33, these two proteins probably use
similar intracellular sorting pathways. Navarro and coworkers
(2004) reported that CymRSV p33 localized exclusively to
peroxisomes when expressed on its own in wild-type S. cerevi-
siae but was localized to ER in a yeast mutant lacking per-
oxisomes as a result of a disruption in the gene encoding the
PMP receptor/chaperone Pex19p. The authors suggested that
CymRSV p33 accumulated in ER in Pex19 mutant cells either
because ER served as a preperoxisome sorting site or because,
in the absence of peroxisomes, sorting to ER occurred simply by
default, as is commonly observed for yeast PMPs (Hettema et al.,
2000). We favor the latter explanation and also propose that
differences observed in sorting p33 in plant and yeast cells are
attributable to differences in the biogenetic relationship between
ER and peroxisomes in these two organisms (reviewed in
Trelease and Lingard, 2005). Thus, CymRSV p33 might not
target to ER from peroxisomes in yeast simply because yeast do
not possess this sorting pathway or because the plant viral
protein cannot use the yeast variant of this pathway.
Properties of the p33 Peroxisomal Membrane
Targeting Signal
A comprehensive analysis of the targeting information in p33
revealed that at least three distinct regions within the N-terminal
half of the protein (i.e., -K11K12-, -K76R77R78R80-, and -R124K129
K130-) constitute the mPTS and were necessary for its initial
sorting from sites of syntheses in the cytosol to peroxisomes
(Figures 7 and 8). One of these regions, -K76R77R78R80-, along
with a C-terminal region (-R213R214R215R216-), were previously
proposed to be the targeting elements responsible for the sorting
of CymRSV p33 to peroxisomes in yeast (Navarro et al., 2004).
However, neither of these regions was demonstrated experi-
mentally to be necessary and sufficient for the peroxisomal
targeting of full-length CymRSV p33, although substitution of the
-R213R214R215R216- with Ala residues prevented peroxisomal
targeting of a minimal CymRSV p33-GFP fusion protein (Navarro
et al., 2004). In our study, gain-of-function and loss-of-function
experiments demonstrated that the C-terminal half of TBSV p33
possessed no peroxisomal targeting information (Figure 7).
Although the relative position (and number) of targeting elements
within TBSV and CymRSV p33 appear to be inconsistent, these
types of mutagenic studies must be interpreted carefully. Aber-
rant protein folding and/or function, rather than disruptions in key
components of peroxisomal targeting signals, can result in
subcellular mislocation. This caveat is especially relevant with
respect to the putative C-terminal targeting element within
CymRSV p33 (Navarro et al., 2004), as this and adjacent regions
of the protein are likely involved in RNA binding (Panaviene et al.,
2003) and p33–p33/p92 protein–protein interactions (Rajendran
and Nagy, 2004).
The mPTS of p33, like most mPTSs, is relatively long, consist-
ing of the N-terminal 156 amino acid residues, including the three
proposed targeting regions and both putative TMDs. All of the
p33-CAT fusion proteins that contained shorter fragments of the
p33 N terminus were either not sorted to peroxisomes or in-
efficiently sorted to peroxisomes (i.e., partiallymislocalized to the
cytosol). Furthermore, disruptions to any of the individual target-
ing regions within the mPTS resulted in p33 being partially
3526 The Plant Cell
mislocalized to the cytosol or pER (Figures 8B and 8C) as well as
inactive in terms of its replicase function (Figure 9). Thus, the
three targeting regions and two TMDswithin themPTS appear to
cooperate to ensure the fidelity of the complexmultistep process
involved in the correct targeting, membrane integration/assem-
bly, and functionality of p33, as proposed previously for the role
of multiple targeting elements in the biogenesis of other PMPs
(Jones et al., 2001; Wang et al., 2001; Murphy et al., 2003).
p33 Possesses a pER Targeting Signal That Resembles an
Arg-Based Motif Responsible for the COPI-Dependent
Retrieval of Resident ER Membrane Proteins
In addition to a mPTS, p33 possesses a pER targeting signal
consisting, in part, of a pair of N-terminal positively charged
residues (-K5R6-). Extensivemutational analysis revealed at least
two important functional properties of the p33 pER targeting
signal. First, the signal overlaps with the N-terminal peroxisome
targeting element whose core component includes the -K11K12-
region, as sorting of p33-K5R6DG to peroxisomes was inefficient
(Figures 8B and 8C). Second, the pER targeting signal functions
from either the cytosol or peroxisomes, but it does so in
a context-dependent manner. For instance, in the case of p33
1–75-CAT, the pER targeting signal was sufficient for sorting
to pER directly from the cytosol, whereas in the context of
full-length p33, the protein was first sorted to peroxisomes (via
the mPTS) before the signal could redirect it to the pER. This
also explains why mutations to any one or more of the three
peroxisomal targeting elements in p33 resulted in the modified
protein(s) being partially localized to pER after only 4 h of
expression (Figure 8), yet wild-type p33 localized exclusively to
peroxisomes at the same time point; these mutations diminish
the overall efficiency of the mPTS and thereby allow the pER
targeting signal to function outside its normal context and
mediate sorting directly from the cytosol to pER. Such a contex-
tual dependence of the pER targeting signal makes it difficult to
elucidate how it functions in an integrative manner with the
protein’smPTS, as noted previously for the overlapping pER (ER)
targeting signal and mPTS in APX (Mullen and Trelease, 2000)
and Pex15p (Elgersma et al., 1997).
To date, several targeting signals responsible for the localiza-
tion of membrane proteins to the ER have been identified,
including cytosolically exposed C-terminal dilysines and aro-
matic amino acid–enriched motifs and N-terminal or internally
positioned Arg residues (Pelham, 2000). In each case, these
targeting signals function in the retrieval of membrane proteins
from the Golgi back to the ER by serving as recognition sites for
specific cytosolic receptor factors, includingCOPI (Aniento et al.,
2003). Because a vesicle transport pathway is most likely re-
sponsible for sorting p33 from peroxisomes to pER, the p33 pER
targeting signal might function in a manner similar to those
involved in COPI-directed transport. There are several striking
similarities between the properties of the -K5R6- region in p33
and the Arg-based ER retrieval motif, a position-independent
signal with a flexible consensus sequence (e.g., -R-R/K- and
-R/K-X-R-) that functions in a COPI-dependent manner in evolu-
tionarily diverse organisms (Schutze et al., 1994; Zerangue et al.,
2001; Yuan et al., 2003). Consistent with these observations, we
showed that replacement of -K5- with -R- in p33, yielding a
prototypic diarginine ER retrieval motif, preserved its sorting to
pER (see Supplemental Figure 3A online). Furthermore, substi-
tution of the N terminus of Arabidopsis a-glucosidase I, a type II
membrane protein that possessed a diarginine ER retrieval motif
(Gillmor et al., 2002), with the N terminus of p33, including the
-K5R6- region, preserved the ER localization of an a-glucosidase
I–GFP fusion protein (see Supplemental Figures 3B and 3C
online). COPI also appears to be involved in the sorting of p33
from peroxisomes to pER, because Arf1Q71L caused coex-
pressed p33 to accumulate exclusively in (aggregated) perox-
isomes (Figure 4B). These results suggest that the Arf1 mutation
prevented the outward vesiculation of the peroxisomal boundary
membrane, as expected based on the established role of this
protein in Golgi-derived vesicle budding (Aniento et al., 2003).
The implication that Arf1 and COPI are involved in peroxisome
vesiculation and peroxisome-to-pER sorting of p33 is not without
precedent. Rat Pex11p contains a C-terminal dilysine ER retrieval
motif that can bind COPI, and although overexpression of Pex11p
causes a proliferation of peroxisomes, cells with defects in the
coatomer vesicle coat possess peroxisomes with pronounced
morphological changes (e.g., tubulation and elongation), consis-
tent with impairment in peroxisome vesiculation (Passreiter et al.,
1998). These data led the authors to propose a dynamicmodel for
the role of Pex11p and COPI in peroxisomal vesiculation in which
the recruitment of COPI to the peroxisomemembrane ismediated
by Pex11p and probably other peroxisomal components (Anton
et al., 2000). Nascent peroxisome-derived vesicles either form into
new mature peroxisomes or function in the return of ER resident
proteins and/or peroxisomal substrates that previously escaped
from the ER via ER-derived vesicles.
Modifications and extensions of the Anton et al. (2000) model
are necessary to incorporate new and previous data on the
possible roles of p33 during pMVB formation. Nascent p33 in
TBSV-infected cells is targeted directly from the cytosol to
peroxisome membranes (via a mPTS), where it is assembled
along with p92 (Rajendran and Nagy, 2004) and viral RNA
(Panaviene et al., 2003) to form a functional replication complex.
This interaction of p33 and p92, and possibly other viral and/or
host cell factors, leads to the inward vesiculation of the perox-
isomal boundary membrane during the initial stages of pMVB
biogenesis. Because the amount of p33 in infected cells is
;20-fold higher than that of p92 (K.B. Scholthof et al., 1995),
excess p33 could also function in a manner similar to that pro-
posed for Pex11p, mediating COPI-dependent budding of the
peroxisomal boundary membrane. The resulting peroxisome-
derived vesicles containing p33 (and other peroxisomal sub-
strates such as early peroxins that function at steady state in the
ER [e.g., Arabidopsis Pex10p; Flynn et al., 2005]) would then be
transported to pER, where, by some unknown means, the viral
protein would modulate the trafficking (via pER-derived vesicles)
of additional membrane constituents required for the increase in
surface area during pMVB biogenesis. Although p33 did not
accumulate in pER in TBSV-infected cells under steady state
conditions (Figure 2B), disruption of the pER targeting signal in
p33 (and p92) in the context of the full-length TBSV genome
resulted in the virus being inactive in terms of its replicase
function (Figures 9B and 9C). Thus, the sorting of p33 to pER
Trafficking of Tomato Bushy Stunt Virus p33 3527
appears to be essential for viral replication, a premise consistent
with electron micrographs of TBSV-infected plant cells showing
pMVBs frequently connected to ER strands that were previously
suggested to be involved in pMVB biogenesis (Martelli et al.,
1988) (Figure 5C). It is possible that, similar to mammalian
Pex11p (Passreiter et al., 1998), only a small amount of p33 is
sorted from peroxisomes to pER and/or that p33 is readily
incorporated into nascent pER-derived vesicles and, thus,
localizedmostly at steady state in pMVBs in TBSV-infected cells.
Although this is a hypothetical model, the mechanism is
appealing because it incorporates pertinent research results
and interpretations of the current working models for the forma-
tion of pMVBs and peroxisome biogenetic pathways in diverse
organisms, particularly in plants. Properly conceived and exe-
cuted experiments are now needed to help resolve the role(s) of
p33 in the context of the wild-type virus and to provide direct
evidence for the intracellular trafficking of this viral protein and
how it functions with, or is modulated by, other proteins in TBSV-
infected cells to control distinct membrane vesiculation events
at peroxisomes/pMVBs and pER, in addition to its role as an
essential component of the viral RNA replication complex. These
types of studies will clearly have an impact on our understand-
ing of the complex means by which p33 along with other TBSV
proteins modify their cellular environment to facilitate viral
replication. Meanwhile, this model not only serves as a basis on
which to analyze unexplored aspects of the cellular processes
underlying TBSV replication, it also reinforces the notion of how
viruses can serve as a useful tool for studying new avenues of
peroxisome biogenesis, including the biogenetic link between
ER and peroxisomes.
METHODS
Recombinant DNA Procedures and Reagents
Standard recombinant DNA procedures were performed as described by
Sambrook et al. (1989). Restriction enzymes and other DNA-modifying
enzymes were purchased from New England Biolabs, and custom syn-
thetic oligonucleotides were obtained from either Invitrogen Life Technol-
ogies, Sigma-Aldrich, or the University of Guelph Laboratory Services.
PCR site-directed mutagenesis of plasmid DNA was performed using
a GeneAmp PCR system 2400 programmable thermal controller (Perkin-
Elmer Canada) and the QuikChange site-directed mutagenesis kit ac-
cording to the manufacturer’s recommendations (Stratagene). Isolation
of DNA fragments and plasmids was performed using Qiagen reagents.
All DNA constructs were confirmed by fluorescent dye-terminator cycle
sequencing at the University of Guelph Molecular Supercentre using an
Applied Biosystems Prism 377 automated sequencer.
Construction of Plasmids
A complete description of all plasmids used in this study and a list of the
sequences of oligonucleotide primers used in plasmid constructions are
available in the online version of this article (see supplemental protocols).
Tobacco BY-2 Cell Cultures, Microprojectile Bombardment, and
Immunofluorescence Microscopy
Tobacco (Nicotiana tabacum) BY-2 suspension-cultured cells were
maintained and prepared for biolistic bombardment as described pre-
viously (Banjoko and Trelease, 1995). Transient transformations were
conducted using 10 mg of plasmid DNA (or 5 mg of each plasmid DNA for
cotransformations) and a PDS1000 biolistic particle delivery system (Bio-
Rad Laboratories). After bombardment, BY-2 cells were incubated at
either 25 or 148C (cold shock) in the dark for 2 to 48 h to allow for
expression of the introduced gene(s) and intracellular protein sorting.
Bombarded cells were then fixed in 4% (w/v) formaldehyde, incubated
with 0.1% (w/v) pectolyase Y-23 (ICN Canada), and permeabilized with
0.3% (v/v) Triton X-100 (Sigma-Aldrich) (Mullen et al., 2001). Cyclohex-
imide (Sigma-Aldrich) was dissolved in BY-2 transformation buffer
(Banjoko and Trelease, 1995) to a final concentration of 100 mM as de-
scribed previously (Imanishi et al., 1998). Briefly, cells at 4 h after bom-
bardment were scraped from filter papers, resuspended in buffer plus
or minus cycloheximide, spread on filter papers, and incubated for an
additional 8 h before fixation.
Fixed and permeabilized BY-2 cells were processed for immunofluo-
rescence microscopy as described by Trelease et al. (1996). Primary and
dye-conjugated secondary antibodies and sources were as follows:
mouse anti-myc (clone 9E10) and rabbit anti-myc IgGs (Berkeley Anti-
body Company); mouse anti-a-tubulin IgGs (clone DM 1A) and rabbit
anti-CAT IgGs (Sigma-Aldrich); mouse anti-CAT hybridoma medium
(provided by S. Subramani, University of California, San Diego); rabbit
anti-cottonseed catalase IgGs (provided by Richard Trelease, Arizona
State University; Kunce et al., 1988); rabbit anti-cottonseed APX IgGs
(provided by R. Trelease; Corpas et al., 1994); mouse monoclonal anti-
tobacco catalase hybridoma medium (Princeton University Monoclonal
Antibody Facility; Chen et al., 1993); mouse anti-maize b-ATPase (pro-
vided by Tom Elthon, University of Nebraska; Luethy et al., 1993); mouse
anti-dsRNA hybridomamedium (clone K2) (provided byWolfgang Nellen,
Kassel University; Schonborn et al., 1991; Bonin et al., 2000); goat anti-
mouse and goat anti-rabbit Alexa 488 IgGs (Molecular Probes); and
goat anti-mouse Cy3 IgGs and goat anti-rabbit rhodamine red-X IgGs
(Jackson ImmunoResearch Laboratories). Rabbit anti-p33 IgGs raised
against a synthetic peptide corresponding to the p33 amino acid sequence
VEPARELKGKDGEDLLTGSR (residues 184 to 203) and purified using
a p33 peptide-Sepharose–linked column were generated by Bethyl Lab-
oratories. For staining ER in BY-2 cells that were also labeled with
antibodies, 5mL of a 1mg/mL stock solution of concanavalin A conjugated
to Alexa 594 or Alexa 394 (Molecular Probes) was added to the cells during
the final 20 min of a 60 min incubation with the secondary antibodies.
Fluorescent images of labeled BY-2 cells were acquired using an
Axioskop 2 MOT epifluorescence microscope (Carl Zeiss) equipped with
a Zeiss 63X Plan Apochromet oil-immersion objective and a Retiga 1300
charged-couple device camera (Qimaging). Images were deconvolved
and/or adjusted for brightness and contrast (Northern Eclipse 7.0; Empix
Imaging) and then composed into figures using Adobe Photoshop 8.0. All
fluorescent images of cells shown in individual figures are representative
of >50 transformed cells from at least two independent transformation
experiments.
Rub-Inoculation and Agrobacterium Infiltration of Tobacco Leaves
Nicotiana benthamiana plants stably transformed with pMAT037/GFP-
SKL were grown in chambers at 218C with a 12-h-light/12-h-dark cycle,
and 5 d before rub-inoculation plants were transferred to a laboratory
bench top with lower light conditions to facilitate the infection process
(Scholthof, 1999). Rub-inoculations (including mock inoculations) were
performed with six- to eight-leaf-stage plants and 5 mg of TBSV RNA
transcripts diluted in 30 mL of RNA inoculation buffer (Scholthof, 1999).
Approximately 5 to8dafter inoculations, tissues fromsystemically infected
upper leaves (Burgyan et al., 1996) were processed for electron micros-
copy.
For transformations of tobacco cv Xanthi using Agrobacterium tume-
faciens infiltration, the binary expression plasmids pMAT037/p33-GFP,
3528 The Plant Cell
pMAT037/p33, pMAT037/GFP-SKL, and pMAT037 were introduced in-
dependently into Agrobacterium (strain LBA4404) using the freeze-thaw
method of Hofgen and Willmitzer (1988). Transformed Agrobacterium
were grown overnight (Hofgen and Willmitzer, 1988), resuspended in
10 mM MgCl2 and 40 mM acetosyringone (Sigma-Aldrich), and then
infiltrated into the upper epidermis of six- to eight-leaf-stage tobacco
plants using a 10-cc syringe as described previously (Grimsley, 1995).
Infiltrated tobacco plants were incubated for 2 or 3 d at the same growth
conditions as those used before the transformation procedure (i.e., 218C
with a 12-h-light/12-h-dark cycle). Approximately 1-cm2 peels of the
lower epidermis of Agrobacterium-infiltrated leaf tissue or 1-cm2 pieces
of whole leaf tissue infiltrated with Agrobacterium were examined using
an epifluorescence microscope or processed for electron microscopy,
respectively.
Electron Microscopy and Immunogold Labeling
TBSVRNA-infiltratedN.benthamiana leaves andAgrobacterium-infiltrated
tobacco leaves were processed for transmission electron microscopy by
fixing tissue pieces in 4% (v/v) glutaraldehyde (Fisher Scientific) and 1%
(v/v) acrolein (Sigma-Aldrich) in 0.05 M KPO4 buffer, pH 7.2, for 2 h in
a vacuum chamber and then at 48C overnight. Samples were then
postfixed overnight at 48C in 1% (w/v) osmium tetroxide (Fisher Scientific),
dehydrated in a graded series of ethanol (50 to 100% [v/v]), and embedded
in Spurr’s medium-grade epoxy (Spurr, 1969). Thin sections were cut with
a glass or diamond knife, mounted on copper grids, and poststained
with 2% (w/v) uranyl acetate and Reynold’s lead citrate (Venable and
Coggeshall, 1965) for 10 and 3min, respectively. All images were acquired
at 80 kV with a Phillips CM10 transmission electron microscope.
Tobacco leaf material was processed for transmission electron mi-
croscopy immunogold labeling as described above except that postfix-
ation in osmium tetroxide was omitted and thin sections were mounted
onto nickel grids. Sections were pretreated with saturated sodium meta-
periodate followed by incubation in a blocking buffer consisting of 33
PBS, 0.05% (w/v) Gly, 0.05% (v/v) Tween 20 (Fisher Scientific), and 0.5%
(w/v) BSA. After incubation in blocking buffer, samples were incubated for
30 min in primary antibodies (rabbit anti-p33 IgGs or preimmune sera)
diluted in blocking buffer, washed a second time in blocking buffer, and
then incubated for 30 min with secondary antibodies (goat anti-rabbit
IgGs conjugated to 5-nm gold particles; Sigma-Aldrich) also diluted in
blocking buffer. Sections were then washed in 13 PBS, washed with
water, air-dried, stainedwith uranyl acetate (Greenwood et al., 2005), and
viewed using a Phillips CM10 transmission electron microscope.
Isolation and Inoculation of BY-2 and Cucumber Protoplasts
Protoplasts prepared from either 3-d-old BY-2 cells (Komoda et al., 2004)
or 6- to 8-d-old cucumber (Cucumis sativus cv Straight 8) cotyledons
(Jones et al., 1990) were quantified by bright-field microscopy using
a hemocytometer. Purified protoplasts were electroporated with 5 mg of
viral RNA transcripts as described previously (White andMorris, 1994). All
TBSV or TBSV-derived transcripts described in Figure 9 were generated
in vitro via transcription of SmaI-linearized template DNAs and using the
Ampliscribe T7 RNA polymerase transcription kit (Epicentre Technolo-
gies) (Oster et al., 1998). Isolated transcripts were quantified spectro-
photometrically and their integrity verified by agarose gel electrophoresis.
After electroporation, BY-2 protoplasts (;1.0 3 106) were pelleted by
centrifugation (500g for 5 min), resuspended in 1 mL of BY-2 culture
medium (Banjoko and Trelease, 1995), and incubated overnight at 268C in
the dark. The next day, protoplasts were centrifuged as described above
and resuspended by gentle pipetting in an equal volume of 23 SDS-
PAGE loading buffer. Aliquots of total protein were separated on 12%
SDS-polyacrylamide gels and electroblotted onto Hybond nitrocellulose
(Amersham Biosciences). Membranes were incubated with anti-p33/p92
antiserum (1:2000) and goat anti-rabbit antiserum conjugated to horse-
radish peroxidase (1:10,000), and immunoreactive proteins were visual-
ized using a Western Lighting kit (Perkin-Elmer) along with a Molecular
Dynamics Storm PhosphorImager (Amersham Biosciences).
Cucumber protoplasts (;33 105) after electroporation were incubated
in a growth chamber under fluorescent lighting at 228C for 24 h and
processed for RNA gel blot analysis as described below.
RNA Gel Blot Analysis of Viral RNAs
RNA was extracted from total nucleic acids harvested from cucumber
protoplasts 24 h after inoculation as described previously (White and
Morris, 1994). Aliquots of isolated RNA were separated in nondenaturing
1.4% (w/v) agarose gels, and viral RNAswere detected by electrophoretic
transfer to nylon (Hybond-N; Amersham Biosciences) followed by in-
cubation with a 32P-end-labeled P9 oligonucleotide probe complemen-
tary to the 39 terminal 23 nucleotides of the TBSV genome (White and
Morris, 1994). Labeled RNAswere visualized and quantified using a phos-
phor imager (Amersham Biosciences).
Accession Numbers
GenBank/EMBL accession numbers for TBSV proteins, including p33
and p92, as well as other proteins described in this study are as follows:
TBSV p33 (NP_062898), TBSV p92 (NP_062897), CymRSV p33
(NP_613261), CNV p33 (AAA42903), Arabidopsis PMP22 (NP_192356),
cottonseed APX (T09845), Arabidopsis Golgi GDP mannose transporter
(NP_178987), pea glutathione reductase (CAA62482),ArabidopsisBS14a
(NP_191376),Arabidopsis a-glucosidase I (NP_176916), andArabidopsis
Arf1 (AAA32729).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Localization of GFP-BS14a inCycloheximide-
Treated Tobacco BY-2 Cells.
Supplemental Figure 2. p33-K5R6K11K12DG Colocalizes with CAT-
APX in Aggregated Peroxisomes, but Not in pER, in Tobacco BY-2
Cells.
Supplemental Figure 3.p33Contains an Arg-Based ERRetrieval Motif.
ACKNOWLEDGMENTS
We thank our colleagues for their generous gifts of antibodies and
plasmids used in this study. We also thank Robb Flynn and Brian Ellis for
providing N. benthamiana and N. tabacum seeds, respectively, Rosa Di
Leo for constructing pRTL2/X-myc, Chris Trobacher for constructing
pRTL2/RFP-HDEL, and Preetinder Dhanoa for constructing monomeric
GFP cassette vectors and pRTL2/GFP-BS14a and for guidance in
experiments involving BS14a. We are also grateful to Kelly Wakeling for
constructing several p33-CAT plasmids, Kimberley Gibson for assis-
tance with sectioning material used for electron microscopy, Ian Smith
for his guidance with Adobe Photoshop, and Baozhong Meng, Annette
Nassuth, John Dyer, Peter Kim, Derek Bewley, and members of our
laboratories for their helpful discussions during the course of this work
and for their comments during the preparation of the manuscript. This
work was supported by grants from the Natural Sciences and Engi-
neering Research Council of Canada (NSERC) to J.S.G., K.A.W., and
R.T.M. K.A.W. holds a Tier II Canada Research Chair, A.W.M. was sup-
ported by an Ontario Graduate Scholarship, M.R.F. was supported by
an NSERC Postgraduate Scholarship, and K.A.W. and R.T.M. are recip-
ients of Ontario Premier’s Research in Excellence Awards.
Trafficking of Tomato Bushy Stunt Virus p33 3529
Received July 19, 2005; revised September 26, 2005; accepted October
12, 2005; published November 11, 2005.
REFERENCES
Aniento, F., Helms, J.B., and Memon, A.R. (2003). How to make
a vesicle: Coat protein-membrane interactions. In The Golgi Appara-
tus and the Plant Secretory Pathway, D.G. Robinson, ed (Oxford, UK:
Blackwell Publishing), pp. 36–62.
Anton, M., Passreiter, M., Lay, D., Thai, T.-P., Gorgas, K., and Just,
W.W. (2000). ARF- and coatomer-mediated peroxisomal vesiculation.
Cell Biochem. Biophys. 32, 27–36.
Appiano, A., Bassi, M., and D’Agostino, G. (1983). Cytochemical and
autoradiographic observations on tomato bushy stunt virus-induced
multivesicular bodies. Ultramicroscopy 12, 162.
Appiano, A., D’Agostino, G., Bassi, M., Barbieri, N., Viale, G., and
Dell’Orto, P. (1986). Origin and function of tomato busy stunt virus-
induced inclusion bodies. II. Quantitative electron microscope auto-
radiography and immunogold cytochemistry. J. Ultrastruct. Mol.
Struct. Res. 97, 31–38.
Baldwin, T.C., Handford, M.G., Yuseff, M.I., Orellana, A., and
Dupree, P. (2001). Identification and characterization of GONST1,
a Golgi-localized GDP-mannose transporter in Arabidopsis. Plant Cell
13, 2283–2295.
Banjoko, A., and Trelease, R.N. (1995). Development and application
of an in vivo plant peroxisome import system. Plant Physiol. 107,
1201–1208.
Bar-Peled, M., and Raikhel, N.V. (1997). Characterization of AtSEC12
and AtSAR1. Proteins likely involved in endoplasmic reticulum and
Golgi transport. Plant Physiol. 114, 315–324.
Bleve-Zacheo, T., Rubino, L., Melillo, M.T., and Russo, M. (1997). The
33 K protein encoded by cymbidium ringspot virus localizes to
modified peroxisomes of infected cells and of uninfected transgenic
plants. J. Plant Pathol. 79, 197–202.
Boevink, P., Martin, B., Oparka, K., Santa Cruz, S., and Hawes, C.
(1999). Transport of virally expressed green fluorescent protein
through the secretory pathway in tobacco leaves is inhibited by
cold shock and brefeldin A. Planta 208, 392–400.
Bonin, M., Oberstrab, J., Luckas, N., Ewert, K., Oesterschulze, E.,
Kassing, R., and Nellen, W. (2000). Determination of preferential
binding sites for anti-dsRNA antibodies on double-stranded RNA by
scanning force microscopy. RNA 6, 563–580.
Brosius, U., Dehmel, T., and Gartner, J. (2002). Two different targeting
signals direct human peroxisomal membrane protein 22 to perox-
isomes. J. Biol. Chem. 277, 774–784.
Burgyan, J., Rubino, L., and Russo, M. (1996). The 59-terminal region
of a tombusvirus genome determines the origin of multivesicular
bodies. J. Gen. Virol. 77, 1967–1974.
Chen, Z., Silva, H., and Klessig, D.F. (1993). Active oxygen species in
the induction of plant systemic acquired resistance by salicylic acid.
Science 262, 1883–1886.
Chew, O., Rudhe, C., Glaser, E., and Whelan, J. (2003). Character-
ization of the targeting signal of dual-targeted pea glutathione re-
ductase. Plant Mol. Biol. 53, 341–356.
Corpas, F.J., Bunkelmann, J., and Trelease, R.N. (1994). Identification
and immunochemical characterization of a family of peroxisome
membrane proteins (PMPs) in oilseed glyoxysomes. Eur. J. Cell Biol.
65, 280–290.
Denecke, J. (2003). Retrograde transport from the Golgi. In The Golgi
Apparatus and the Plant Secretory Pathway, D.G. Robinson, ed
(Oxford, UK: Blackwell Publishing), pp. 90–99.
Elgersma, Y., Kwast, L., van den Berg, M., Snyder, W.B., Distel, B.,
Subramani, S., and Tabak, H.F. (1997). Overexpression of Pex15p,
a phosphorylated peroxisomal integral membrane protein required for
peroxisome assembly in S. cerevisiae, causes proliferation of the
endoplasmic reticulum membrane. EMBO J. 16, 7326–7341.
Fabian, M.R., and White, K.A. (2004). 59-39 RNA-RNA interaction
facilitates cap- and poly(A) tail-independent translation of tomato
bushy stunt virus mRNA: A potential common mechanism for Tom-
busviridae. J. Biol. Chem. 279, 28862–28872.
Farre, J.C., and Subramani, S. (2004). Peroxisome turnover by micro-
pexophagy: An autophagy-related process. Trends Cell Biol. 14,
515–523.
Flynn, C.R., Heinze, M., Schumann, U., Gietl, C., and Trelease, R.N.
(2005). Compartmentalization of the plant peroxin, AtPex10p, within
subdomain(s) of ER. Plant Sci. 168, 635–652.
Gillmor, C.S., Poindexter, P., Lorieau, J., Palcic, M.M., and Somerville,
C. (2002). Alpha-glucosidase I is required for cellulose biosynthesis
and morphogenesis in Arabidopsis. J. Cell Biol. 156, 1003–1013.
Greenwood, J.S., Helm, M., and Gietl, C. (2005). Ricinosomes and
endosperm transfer cell structure in programmed cell death of the
nucellus during Ricinus seed development. Proc. Natl. Acad. Sci. USA
102, 2238–2243.
Grimsley, N. (1995). Agroinfection. Methods Mol. Biol. 44, 325–342.
Hettema, E.H., Girzalsky, W., van Den Berg, M., Erdmann, R., and
Distel, B. (2000). Saccharomyces cerevisiae pex3p and pex19p are
required for proper localization and stability of peroxisomal membrane
proteins. EMBO J. 19, 223–233.
Hofgen, R., and Willmitzer, L. (1988). Storage of competent cells for
Agrobacterium transformation. Nucleic Acids Res. 16, 9877.
Imanishi, S., Hashizume, K., Nakakita, M., Kojima, H., Matsubayashi,
Y., Hashimoto, T., Sakagami, Y., Yamada, Y., and Nakamura, K.
(1998). Differential induction by methyl jasmonate of genes encoding
ornithine decarboxylase and other enzymes involved in nicotine
biosynthesis in tobacco cell cultures. Plant Mol. Biol. 38, 1101–1111.
Jedd, G., and Chua, N.H. (2002). Visualization of peroxisomes in living
plant cells reveals acto-myosin-dependent cytoplasmic streaming
and peroxisome budding. Plant Cell Physiol. 43, 384–392.
Jones, J.M., Morrell, J.C., and Gould, S.J. (2001). Multiple distinct
targeting signals in integral peroxisomal membrane proteins. J. Cell
Biol. 153, 1141–1149.
Jones, R.W., Jackson, A.O., and Morris, T.J. (1990). Defective-
interfering RNAs and elevated temperatures inhibit replication of tomato
bushy stunt virus in inoculated protoplasts. Virology 176, 539–545.
Karnik, S.K., and Trelease, R.N. (2005). Arabidopsis thaliana peroxin
16 (AtPex16p) coexists at steady state in peroxisomes and endo-
plasmic reticulum. Plant Physiol. 318, 1967–1981.
Komoda, K., Naito, S., and Ishikawa, M. (2004). Replication of plant
RNA virus genomes in a cell-free extract of evacuolated plant
protoplasts. Proc. Natl. Acad. Sci. USA 101, 1863–1867.
Kunce, C.M., Trelease, R.N., and Turley, R.B. (1988). Purification and
biosynthesis of cottonseed (Gossypium hirsutum L.) catalase. Bio-
chem. J. 251, 147–155.
Lisenbee, C.S., Heinze, M., and Trelease, R.N. (2003a). Peroxisomal
ascorbate peroxidase resides within a subdomain of rough endoplasmic
reticulum in wild-type Arabidopsis cells. Plant Physiol. 132, 870–882.
Lisenbee, C.S., Karnik, S.K., and Trelease, R.N. (2003b). Overexpres-
sion and mislocalization of a tail-anchored GFP redefines the identity
of peroxisomal ER. Traffic 4, 491–501.
Luethy, M.H., Horak, A., and Elthon, T.E. (1993). Monoclonal anti-
bodies to the a- and b-subunits of the plant mitochondrial F1-ATPase.
Plant Physiol. 101, 931–937.
Martelli, G.P., Gallitelli, D., and Russo, M. (1988). Tombusviruses. In
The Plant Viruses. Vol. 3. Polyhedral Virions with Monopartite RNA
Genomes, R. Koenig, ed (New York: Plenum Press), pp. 13–72.
3530 The Plant Cell
Mullen, R.T., Lisenbee, C.S., Flynn, C.R., and Trelease, R.N. (2001).
Stable and transient expression of chimeric peroxisomal membrane
proteins induces an independent ‘‘zippering’’ of peroxisomes and an
endoplasmic reticulum subdomain. Planta 213, 849–863.
Mullen, R.T., Lisenbee, C.S., Miernyk, J.A., and Trelease, R.N. (1999).
Peroxisomal membrane ascorbate peroxidase is sorted to a membra-
nous network that resembles a subdomain of the endoplasmic
reticulum. Plant Cell 11, 2167–2185.
Mullen, R.T., and Trelease, R.N. (2000). The sorting signals for
peroxisomal membrane-bound ascorbate peroxidase are within its
C-terminal tail. J. Biol. Chem. 275, 16337–16344.
Murphy, M.A., Phillipson, B.A., Baker, A., and Mullen, R.T. (2003).
Characterization of the targeting signal of the Arabidopsis 22-kD
integral peroxisomal membrane protein. Plant Physiol. 133, 813–828.
Nagata, T., Nemoto, Y., and Hasezawa, S. (1992). Tobacco BY-2 cell
line as the ‘‘Hela’’ cell in the cell biology of higher plants. Int. Rev.
Cytol. 132, 1–30.
Navarro, B., Rubino, L., and Russo, M. (2004). Expression of the
Cymbidium ringspot virus 33-kilodalton protein in Saccharomyces
cerevisiae and molecular dissection of the peroxisomal targeting
signal. J. Virol. 78, 4744–4752.
Nito, K., Yamaguchi, K., Kondo, M., Hayashi, M., and Nishimura, M.
(2001). Pumpkin peroxisomal ascorbate peroxidase is localized on
peroxisomal membranes and unknown membranous structures. Plant
Cell Physiol. 42, 20–27.
Oster, S.K., Wu, B., and White, K.A. (1998). Uncoupled expression of
p33 and p92 permits amplification of tomato bushy stunt virus RNAs.
J. Virol. 72, 5845–5851.
Panavas, T., Hawkins, C.M., Panaviene, Z., and Nagy, P.D. (2005).
The role of the p33:p33/p92 interaction domain in RNA replication and
intracellular localization of p33 and p92 proteins of cucumber necrosis
tombusvirus. Virology 338, 81–95.
Panaviene, Z., Baker, J.M., and Nagy, P.D. (2003). The overlapping
RNA-binding domains of p33 and p92 replicase proteins are essential
for tombusvirus replication. Virology 308, 191–205.
Passreiter, M., Anton, M., Lay, D., Frank, R., Harter, C., Wieland,
F.T., Gorgas, K., and Just, W.W. (1998). Peroxisome biogenesis:
Involvement of ARF and coatomer. J. Cell Biol. 141, 373–383.
Pelham, H.R.B. (2000). Using sorting signals to retain proteins in
endoplasmic reticulum. Methods Enzymol. 327, 279–283.
Pepperkok, R., Whitney, J.A., Gomez, M., and Kreis, T.E. (2000).
COPI vesicles accumulating in the presence of a GTP restricted Arf1
mutant are depleted of anterograde and retrograde cargo. J. Cell Sci.
113, 135–144.
Phillipson, B.A., Pimpl, P., daSilva, L.L., Crofts, A.J., Taylor, J.P.,
Movafeghi, A., Robinson, D.G., and Denecke, J. (2001). Secretory
bulk flow of soluble proteins is efficient and COPII dependent. Plant
Cell 13, 2005–2020.
Purdue, P.E., and Lazarow, P.B. (2001). Peroxisome biogenesis. Annu.
Rev. Cell Dev. Biol. 17, 701–752.
Rajendran, K.S., and Nagy, P.D. (2004). Interaction between the
replicase proteins of tomato bushy stunt virus in vitro and in vivo.
Virology 326, 250–261.
Rubino, L., and Russo, M. (1998). Membrane targeting sequences in
tombusvirus infections. Virology 252, 431–437.
Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular
Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor, NY:
Cold Spring Harbor Laboratory Press).
Scholthof, H.B. (1999). Rapid delivery of foreign genes into plants by
direct rub-inoculation with intact plasmid DNA of a tomato bushy stunt
virus gene vector. J. Virol. 73, 7823–7829.
Scholthof, H.B., Scholthof, K.B., Kikkert, M., and Jackson, A.O.
(1995). Tomato bushy stunt virus spread is regulated by two nested
genes that function in cell-to-cell movement and host-dependent
systemic invasion. Virology 213, 425–438.
Scholthof, K.B., Scholthof, H.B., and Jackson, A.O. (1995). The
tomato bushy stunt virus replicase proteins are coordinately ex-
pressed and membrane associated. Virology 208, 365–369.
Schonborn, J., Oberstrass, J., Breyel, E., Tittgen, J., Schumacher,
J., and Lukacs, N. (1991). Monoclonal antibodies to double-stranded
RNA as probes of RNA structure in crude nucleic acid extracts.
Nucleic Acids Res. 19, 2993–3000.
Schutze, M.P., Peterson, P.A., and Jackson, M.R. (1994). An
N-terminal double-arginine motif maintains type II membrane proteins
in the endoplasmic reticulum. EMBO J. 13, 1696–1705.
Spurr, A.R. (1969). A low-viscosity epoxy resin embedding medium for
electron microscopy. J. Ultrastruct. Res. 26, 31–43.
Takeuchi, M., Ueda, T., Yahara, N., and Nakano, A. (2002). Arf1
GTPase plays roles in the protein traffic between the endoplasmic
reticulum and the Golgi apparatus in tobacco and Arabidopsis
cultured cells. Plant J. 31, 499–515.
Trelease, R.N. (2002). Peroxisomal biogenesis and acquisition of
membrane proteins. In Plant Peroxisomes: Biochemistry, Cell Biology
and Biotechnological Applications, A. Baker and I. Graham, eds (Dor-
drecht, The Netherlands: Kluwer Academic Publishers), pp. 305–337.
Trelease, R.N., Lee, M.S., Banjoko, A., and Bunkelmann, J. (1996).
C-terminal polypeptides are necessary and sufficient for in vivo target-
ing of transiently-expressed proteins to peroxisomes in suspension-
cultured plant cells. Protoplasma 195, 156–167.
Trelease, R.N., and Lingard, M.J. (2005). Participation of the plant ER
in peroxisomal biogenesis. In The Plant Endoplasmic Reticulum (Plant
Cell Monographs), D.G. Robinson, ed (Heidelberg, Germany:
Springer-Verlag), in press.
Uemura, T., Ueda, T., Ohniwa, R., Nakano, A., Takeyasu, K., and Sat,
M.H. (2004). Systematic analysis of SNARE molecules in Arabidopsis:
Dissection of the post-Golgi network in plant cells. Cell Struct. Funct.
29, 49–65.
Venable, J.H., and Coggeshall, R. (1965). Simplified lead citrate stain
for use in electron microscopy. J. Cell Biol. 25, 407–408.
Wang, X., Unruh, M.J., and Goodman, J.M. (2001). Discrete targeting
signals direct Pmp47 to oleate-induced peroxisomes in Saccharomy-
ces cerevisiae. J. Biol. Chem. 276, 10897–10905.
White, K.A., and Morris, T.J. (1994). Nonhomologous RNA recombi-
nation in tombusviruses: Generation and evolution of defective in-
terfering RNAs by stepwise deletions. Virology 68, 14–24.
White, K.A., and Nagy, P.D. (2004). Advances in the molecular biology
of tombusviruses: Gene expression, genome replication and recom-
bination. Prog. Nucleic Acid Res. Mol. Biol. 78, 187–226.
Wroblewski, T., Tomczak, A., and Michelmore, R.W. (2005). Optimi-
zation of Agrobacterium-mediated transient assays of gene expression
in lettuce, tomato and Arabidopsis. Plant Biotechnol. J. 3, 259–273.
Yuan, H., Michelsen, K., and Schwappach, B. (2003). 14-3-3 dimers
probe the assembly status of multimeric membrane proteins. Curr.
Biol. 13, 638–646.
Zerangue, N., Malan, M.J., Fried, S.R., Dazin, P.F., Jan, Y.N., Jan,
L.Y., and Schwappach, B. (2001). Analysis of endoplasmic reticulum
trafficking signals by combinatorial screening in mammalian cells.
Proc. Natl. Acad. Sci. USA 98, 2431–2436.
Trafficking of Tomato Bushy Stunt Virus p33 3531
DOI 10.1105/tpc.105.036350; originally published online November 11, 2005; 2005;17;3513-3531Plant Cell
Andrew W. McCartney, John S. Greenwood, Marc R. Fabian, K. Andrew White and Robert T. MullenPeroxisome-to-Endoplasmic Reticulum Sorting Pathway
Localization of the Tomato Bushy Stunt Virus Replication Protein p33 Reveals a
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