1 Ruhr-Universität Bochum, Institut für Physiologische Chemie,
Medizinische Fakultät, 44780 Bochum, Germany
2 Institut für Toxikologie und Genetik, Forschungszentrum Karlsruhe,
Postfach 3640, 76021 Karlsruhe, Germany
* Author for correspondence (e-mail: nils.johnsson{at}itg.fzk.de)
Accepted 21 May 2003
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Summary |
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Key words: Membrane proteins, Protein interaction, Peroxins, Peroxisome, Protein import, Split-ubiquitin
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Introduction |
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Materials and Methods |
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NUI-PEX3, -PEX4, -PEX15,
-PEX14, -PEX19, -PEX11 and -PEX10 were obtained by fusing a PCR
product covering the respective ORF and approximately 200 bp of 3'
untranslated sequence in frame behind the
PCUP1-NUI module
using the BamHI and a second restriction site.
PCUP1-NUI-PEX1
and -PEX5 contained only the first 442 and 480 bp of the respective
ORFs. Full-length NUI-PEX1 and
NUI-PEX5 were created via homologous
recombination by transforming the yeast with the EcoRI
(PEX1) or Acc651 (PEX5) cut plasmids
(Dünnwald et al., 1999).
NUI-PEX12 and
NUI-PEX13 contained an additional
sequence at their 3' end encoding the HA epitope. The linker sequence
connecting NUI and the PEX gene reads:
GGG ATC CCT GGG GAT XXX, with the BamHI site underlined
and XXX denoting the second codon of the attached PEX gene.
PEX12-NUI and
PEX22-NUI were obtained by inserting the
respective ORF between the sequences of the
PCUP1-promoter and of
NUI using the EcoRI and SalI
restriction sites. The sequence between PEX22 and
NUI reads: XXX GGG TCG ACC GGC GGT
ATG. XXX denotes the last codons of PEX22/PEX12 and ATG the first
codon of NUI. The SalI is underlined. PEX4-6HA and
PEX10-6HA were constructed by cutting the respective ORF after PCR
amplification with EcoRI and SalI and inserting the fragment
between the PCUP1-promoter and a sequence encoding six
consecutive HA-epitopes on a pRS313 vector (N. Lewke, Köln, Germany). The
sequences encoding SKL and SSS were attached via a PCR in frame behind the
PCUPI-NUI-HA module
(Wittke et al., 1999
).
NUI-PEX1 and
NUI-PEX5 resided on the PRS304 vector.
All other NUI constructs resided on a PRS314
vector. PEX4-, PEX10, PEX11-, PEX12- and PEX22-9MYC
were obtained by integrative insertion according to Knop et al.
(Knop et al., 1999
). All
oligonucleotides were obtained from Metabion (Martinsried, Germany).
Additional information on the generation of Nub-
and Cub-constructs can be obtained on
request.
Deletion of ORFs
The ORFs of the PEX genes were deleted from the strain JD53
according to Güldener et al.
(Güldener et al., 1996).
Transformed yeast cells were selected for kanr integration by
Geneticin (Life Technologies, Paisley, Scotland). The deletions were verified
by diagnostic PCR and the inability of the cells to grow on media containing
oleate as the sole carbon source. See Table
1 for a list of yeast strains used.
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Growth and interaction assays
The assays used yeast-rich (YPD) and synthetic minimal media with 2%
dextrose (SD) and followed standard protocols. Transformed JD53 cells were
grown at 30°C on selective media containing uracil. Cells were suspended
and diluted in sterile water to an OD600 of 1 µl and 4 µl,
and 4 µl of tenfold serial dilutions were spotted on agar plates, selecting
for the presence of the fusion constructs but lacking uracil (SD-Ura) or
containing both 1 mg/ml 5'-Fluororotic acid and 50 µg/ml uracil (FOA;
WAK-Chemie, Bad-Soden, Germany). The same dilutions were spotted on plates
containing uracil to check for cell numbers. The plates were incubated at
30°C for 2-4 days unless otherwise stated.
For testing the growth on oleate, cells were first incubated on SD medium containing reduced concentrations of glucose (0.3%). After 2-3 days at 30°C, cells were transferred onto oleate media containing 1.26% oleic acid and 5% Tween® 40 (palmitate) as sole carbon sources. The plates were incubated at 30°C for 7 days or longer. The ability of the cells to grow and the appearance of the characteristic clearances around the colonies were used to determine the functionality of the fusion proteins.
GFP-SKL import assay
Wild-type and mutant JD53 cells were transformed with pEW88, a plasmid
coding for GFP-SKL (courtesy of Ben Distel and Ewald Hettema, Amsterdam, The
Netherlands). Cells were assayed for GFPSKL import into peroxisomes by the
appearance of the characteristic punctuated, intracellular fluorescence under
a Leica DMRB Microscope equipped with a Xenon-75W-GFP/XBO75 lamp and an L4
filter with a cut-off at 500 nm (Leica, Solms, Germany).
Co-immunoprecipitation of peroxisomal membrane proteins
A 300 ml culture of yeast cells was grown to OD600 0.8-1.0 under
aeration at 30°C. Cells were collected at 3000 g and
washed twice with distilled water at room temperature. The cells were
resuspended in 3 ml ice-cold IP-buffer (50 mM Tris-HCl, pH 7.5; 50 mM NaCl;
0.2% (V/V) Triton X-100; 0.25 mM PMSF; 15 µg/ml Antipain; 1.5 µg/ml
Pepstatin A; 1.0 µg/ml Leupeptin; 0.1 µg/ml Chymostatin) and were broken
up in glass vials by the addition of 3 g acid-washed glass beads (425-600
µm) and by vigorous vortexing (15x30 seconds with 30-second intervals
on ice). The suspension was centrifuged for 15 minutes at 5000
g at 4°C and the supernatant was centrifuged again for 30
minutes at 4°C and 17,500 g. Five microlitres of the
primary antibody were added to the supernatant and incubated at 4°C for 60
minutes. Twenty-five microlitres of protein A-sepharose beads (Roche
Diagnostics, Mannheim, Germany) were added to each vial and incubated for at
least 2 hours at 4°C. The beads were washed three times with 2 ml
IP-buffer and finally boiled in 80 µl SDS-sample buffer.
Chemical cross-linking
A 500 ml yeast culture was incubated under aeration at 30°C to
OD600 1.5-2.0. Cells were collected by centrifugation at 1500
g and 4°C for 5 minutes. One gram of cells was defined as
one volume (Vol) for all subsequent steps. The cell pellet was washed in 2-4
Vol ice-cold distilled water and resuspended in 1 Vol Lyticase-buffer (50 mM
KH2PO4, pH 7.5; 10 mM MgCl2; 1 M Sorbitol; 1
mM dithiothreitol (DTT)) containing 30 mM DTT. After incubation at room
temperature for 15 minutes the cells were pelleted by centrifugation for 5
minutes at 1500 g and 4°C and resuspended in 3 Vol
Lyticase-buffer. 200 U Lyticase (Sigma, Deisenhofen, Germany) per Vol were
added and the cell suspension was incubated for 40 minutes at 30°C under
slow shaking (50 rpm). Spheroblasting was confirmed by the lysis-in-water
test of small samples. Cells were centrifuged for 5 minutes at 1500
g and 4°C and resuspended in 10 Vol ice-cold cross-linking
buffer (100 mM KH2PO4, pH 7.5; 1 mM EDTA; 0.25 mM PMSF;
15 µg/ml Antipain; 1.5 µg/ml Pepstatin A; 1.0 µg/ml Leupeptin; 0.1
µg/ml Chymostatin), and washed twice. The cells were resuspended in 1 Vol
ice-cold cross-linking buffer and were lysed by ten strokes with a glas
douncer. DSP (Pierce, USA) in DMSO was added to a final concentration of 200
µg/ml and the extracts were incubated for 30 minutes at 23°C. After
quenching by the addition of glycine to 20 mM the cross-linked proteins were
coprecipitated as decribed above.
SDS-PAGE (polyacrylamide gel electrophoresis), transfer onto nitrocellulose
and immunodetection of the transferred proteins was performed as described
previously (Wittke et al.,
2002). For sequential detection of proteins, membranes were
stripped with 2% SDS and 100 mM ß-mercaptoethanol for 30 minutes at
55°C.
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Results |
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The localization and assumed topologies of the Nub- and
Cub-labeled fusion proteins that were used in this study are shown
in Fig. 1B. Excluded from our
study were those peroxins that are known to reside in the lumen of the
peroxisome or that are exclusively involved in the import of proteins carrying
the PTS2 import signal. Also not considered as Nub fusions were
those peroxins whose N-termini had been shown or strongly suspected to point
into the matrix of the organelle. To control the specificity of the assay, we
measured the reactions between Nub-Pex11p and the
Cub-RUra3p-labeled Pex2p, Pex10p and Pex12p. Pex11p is a
peroxisomal membrane protein that does not participate in the import of matrix
proteins (Erdmann and Blobel,
1995; Li and Gould,
2002
). We therefore assumed that Pex11p is not permanently
integrated into any of the peroxin import complexes and should therefore serve
as a valid control. To verify the correct localization of the
Cub-modified peroxins, we measured their interactions with a set of
Nub-labeled membrane proteins known to reside in other compartments
of the cell (Wittke et al.,
1999
).
RING-finger-dependent interaction between Pex12p and Pex10p, Pex13p
and Pex15p
We attached the Cub-RUra3p module to the C-terminus of Pex12p to
construct Pex12-Cub-RUra3p. Yeast cells bearing
Pex12-Cub-RUra3p were transformed with the indicated
Nub-fusions and the transformants were spotted onto plates lacking
uracil or containing 5-FOA (Fig.
2A,B). In contrast to the other cotransformants, the cells
containing Nub-Pex19p, Nub-Pex15p, Nub-Pex13p
or Nub-Pex10p do not grow on SD-ura, but grow on 5-FOA instead. As
we expected from their different roles in peroxisome biogenesis,
Pex12-Cub-RUra3p did not interact with Nub-Pex11p in our
assay. To confirm that the non-growth/growth of the cells on SD-ura/5-FOA
indeed reflects the interaction-depended reassociation of the attached
Nub and Cub, we performed the same assay in cells
lacking a functional N-end rule pathway. Cells lacking the recognition
component of the N-end-rule pathway (UBRI) will not degrade the
cleaved RUra3p (Varshavsky et al.,
2000). Consequently, the growth of the cells on media containing
5-FOA or lacking uracil should not be influenced by the efficiency of the
Nub-Cub reassociation of the co-expressed fusion
proteins. Accordingly, and in contrast to the isogenic wild-type cells, we
observed good growth on SD-ura of the cells lacking UBR1,
irrespective of the identity of their expressed Nub and
Cub fusion proteins (Fig.
2D). We conclude that Pex12p interacts with Pex10p, Pex15p, Pex19p
and Pex13p. To estimate the contribution of the RING-finger to these
interactions, we tested a Cub-RUra3p fusion of Pex12p in which the
entire C-terminal sequence (residues 320-399) was deleted
(Pex12
C-Cub-RUra3p). This deletion abolishes almost all
interaction signals, leaving Nub-Pex19p as the only interaction
partner of Pex12
C-Cub-RUra3p
(Fig. 3). Besides pointing to
the crucial role of the C-terminal domain of Pex12p for its interaction with
Pex10p, Pex13p and Pex15p, this experiment again confirms that the measured
interactions of the full-length Pex12p are specific.
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Pex10p is in close proximity to Pex4p
The interaction profile of Pex10-Cub-RUra3p differs in two
aspects from the interaction profile of Pex12-Cub-RUra3p (compare
Fig. 4A,B with
Fig. 2A,B). Apart from the
interactions that were already detected for Pex12p, we observed an additional
close proximity between Pex10p and Pex4p, as well as a Pex10p-Pex10p
interaction. The weak interaction signal that is observed between
Nub-Pex3p and Pex10-Cub-RUra3p on 5-FOA is not above the
background that is defined in this experiment by the weak interaction signal
of the cells expressing Pex10-Cub-RUra3p and Nub-Tom22p.
We conclude that Pex10p and Pex12p share most of their interaction partners,
except for Pex4p, which forms a unique interaction with Pex10p. Pex4p is a
ubiquitin conjugating enzyme and its measured proximity to Pex10p points to a
functional involvement of the RING-finger of Pex10p in a Pex4p-mediated
ubiquitylation reaction. Unfortunately, the influence of the role of the
RING-finger of Pex10p on the observed interactions could not be assessed. The
cellular amount of the corresponding Pex10C-Cub-RUra3p was
already so low that the growth of the cells on SD-ura or 5-FOA could no longer
serve as an indicator of interaction. To independently confirm some of the
newly identified protein interactions of Pex10p (see
Fig. 4A,B and
Table 2), we performed
co-immunoprecipitations between the HA-tagged Pex10p and the MYC-tagged
versions of Pex10p, Pex12p, Pex4p and Pex22p. A MYC-tagged Pex11p was
introduced as a control for evaluating the specificity of the
immunoprecipitations. Pex22p was included because of its measured interaction
with Pex10p in a strain expressing
Pex22-Cub-RUra3p/Nub-Pex10p
(Table 2). Fig. 4C shows the
co-precipitation of the two differently tagged Pex10p fusions and the
precipitation of Pex12p by Pex10p. MYC-tagged Pex4p and Pex22p co-precipitated
with Pex10p only when the precipitation of Pex4p and Pex22p by the MYC
antibody was followed by detection of the labeled Pex10p with the HA antibody
(Fig. 4D). Changing the order
of the employed antibodies in the experiment did not result in measurable
co-precipitation. Our interpretation is that the binding of Pex10p to Pex4p
and Pex22p is only temporal or so labile that the complexes only partially
survive our immunoprecipitation protocol. The binding of the HA antibody to
the C-terminus of the labeled Pex10p might further weaken an already unstable
interaction. We gained additional confidence in the presence of a Pex4p-Pex10p
complex through its independent detection in a genome-wide two-hybrid
experiment (P. Uetz, personal communication) and a systematic two-hybrid
experiment on peroxins (K. Schulz and R. Erdmann, personal communication).
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Pex2p interacts only with Pex19p in the split-Ub assay
Pex2-Cub-RUra3p as the third RING-finger peroxin displayed a
completely different interaction profile when co-expressed with the same set
of Nub-fusions (Fig.
5A,B). We only detected an interaction between Pex2p and Pex19p.
Nub-Pex13p, -Pex10p and -Pex15p, although interacting with both the
Cub-RUra3p-labeled Pex12p and Pex10p, are not in close proximity to
Pex2-Cub-RUra3p.
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As already observed for the correspondingly modified Pex12p and Pex10p,
Pex2-Cub-RUra3p also does not interact with Nub-Pex11p
(Figs 2,
3,
4,
5). These results are in
agreement with the assumed absence of Pex11p in any of the peroxin complexes.
However, to be considered as a valuable control, we had to confirm that the
lack of Nub-Pex11p interactions are not caused by either the
mislocalization or a low activity of Nub-Pex11p. We therefore
constructed Pex11-Cub-RUra3p and determined its proximity to the
same set of Nub-fusions (Fig.
5C,D). Pex11-Cub-RUra3p does not interact with any of
the other Nub-labeled peroxins except Nub-Pex19p and
Nub-Pex11p. Because Pex11p is known to self-multimerize, our
observation confirms this behavior for the fusion protein in vivo and
validates Nub-Pex11p as a proper control for the split-Ub measured
peroxin interactions (Marshall et al.,
1996).
As expected, Nub-Sec22, Nub-Vam3p or
Nub-Guk1p as representatives of other cellular compartments did not
show close proximity to any of the RING-finger peroxins (Figs
2,
3,
4,
5). Only cells co-expressing
the mitochondrial marker protein Nub-Tom22p together with
Pex10-Cub-RUra3p or Pex12CCub-RUra3p displayed a
weak interaction signal that might point to a certain amount of
mislocalization of either Nub-Tom22p or the
Cub-RUra3p-labeled Pex10p and Pex12
Cp
(Fig. 4 and authors'
unpublished observation).
An extended network of peroxin interactions
To integrate the interactions of the RING-finger peroxins into a larger
context, we tested an extended subset of Cub-RUra3p fusions against
the same set of Nub fusion proteins. The measured interactions
between the different Nub- and Cub-labeled peroxins are
summarized in Table 2 and
Fig. 8. We only interpreted
growth or non-growth on 5-FOA or SD-ura as an indication of interaction if the
signals were significantly stronger than those derived from the co-expression
of Nub-Pex11p or other Nub-fusion proteins that were
introduced as controls. To assist the interpretation of the interaction data,
we measured the functionality of all fusion constructs by complementation
tests of the corresponding deletion strains. Successful complementation was
determined by two criteria: (1) peroxisomal localization of a green
fluorescent protein (GFP) carrying a peroxisome import signal and (2) growth
on oleic acid as the sole carbon source. The results of the complementation
assays are documented in Table
3. We regard the interaction signal between Nub-Tom22p
and Pex13-Cub-RUra3 on 5-FOA plates as a false positive
(Table 2). Because
Pex13-Cub-RUra3p is not functional, the observed interaction might
indicate a high percentage of mislocalization of Pex13-Cub-RUra3p
to the mitochondrion (Tables 2,
3). Pex10-Cub-RUra3p
displays a similar, albeit less extreme, behaviour
(Fig. 4A,B).
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Pex22p is required for the Pex4p-Pex10p interaction
The split-Ub assay measures a close proximity rather than a direct
interaction between the Nub- and Cub-labeled membrane
proteins. Although the measured interactions can be indirect, the technique
requires a relatively close apposition of the two labeled termini of the
proteins. Spatially more distant subunits in a large assembly might therefore
give no or only a very weak interaction signal as
Cub/Nub-modified proteins, whereas a strong interaction
signal indicates a close proximity between the two labeled subunits in this
complex. A split-Ub measured interaction might therefore reflect aspects of
the actual geometry of the complex. This assumption helps to explain why Pex4p
interacts with Pex10p, but not with Pex12p or Pex13p, although Pex10p, Pex12p
and Pex13p were all found to be connected through interactions
(Table 2). As can be seen from
Table 2, Nub-Pex4p
interacts not only with Pex10-Cub-RUra3p, but also with
Pex22-Cub-RUra3p and Pex4-Cub-RUra3p.
Pex22-Cub-RUra3p interacts with Nub-Pex4p,
Nub-Pex10p, Nub-Pex15p and Nub-Pex19p
(Table 2). The interaction
between Nub-Pex10p and Pex22-Cub-RUra3p is only seen on
5-FOA, but could be confirmed by a co-precipitation experiment
(Fig. 4D). To estimate the
importance of Pex22p for the interaction between Pex10p and Pex4p, we measured
the proximity between Nub-Pex4p and Pex10-Cub-RUra3p in
cells lacking Pex22p (Fig. 6).
The absence of Pex22p only impairs the interaction between Pex10p and Pex4p,
whereas the interactions between Pex10p and Pex12p, Pex10p, Pex13p, Pex15p and
Pex19p persist (Fig. 6A,C). The
effect is specific for Pex22p given that the deletion of Pex5p does not affect
the interaction between Pex4p and Pex10p nor any of the other measured
interactions of Pex10p (Fig.
6A,B). It was reported that the concentration of Pex4p is reduced
on deletion of PEX22 in Pichia pastoris
(Koller et al., 1999).
However, our Saccharomyces cerevisiae pex22
cells express
enough Ura3p activity from Pex4-Cub-RUra3p to confer 5-FOA
sensitivity and they express sufficient amounts of Nub-Pex4p to
enable the interaction between Nub-Pex4p and
Pex4-Cub-RUra3p to be measured
(Fig. 6D). The Pex4p-Pex4p
interaction is also detected in wild-type cells and is specific, given that
the endoplasmic reticulum (ER)-based Nub-Ubc6p does not interact
with Pex4-Cub-RUra3p (Fig.
6E). Ubc6p and Pex4p belong to the same family of ubiquitin
conjugating enzymes (Pickart,
2001
). The split-Ub-measured proximity between Nub-and
Cub-labeled Pex4p could be confirmed by the co-precipitation of HA
and MYC-tagged Pex4p from yeast extracts. Successful co-precipitation required
the addition of the chemical crosslink DSP before the incubation with
antibodies (Fig. 6F).
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Interaction assays in the absence of peroxisomal membranes
Pex3p and Pex19p are both crucial for the formation of peroxisomal
membranes. In the absence of either of the two corresponding genes, the cells
are free of any recognizable peroxisomal structures
(Ghaedi et al., 2000;
Matsuzono et al., 1999
;
South and Gould, 1999
). This
feature distinguishes Pex19p and Pex3p from all the other known peroxins,
although recent work revealed remnant membranous structures in
pex3
cells, which are, however, different from those found in
other peroxin deletion strains (Hazra et
al., 2002
). The importance of Pex19p for the biogenesis of the
peroxisome is clearly reflected in our interaction matrix. All
peroxin-Cub-RUra3p fusion proteins show interactions with
Nub-Pex19p, except for Pex4-Cub-RUra3p and
Pex3-Cub-RUra3p (Table
2, Fig. 8). Pex3p
does not share this feature of multiple interactions and only reveals a weak
interaction signal with Pex15p and Pex13p in our assay
(Table 2). To gauge the
importance of Pex3p and thereby the importance of the peroxisomal membrane for
the interactions of the RING-finger peroxins Pex10p and Pex12p, we performed
the interaction assays in cells lacking Pex3p. Nub-Pex10p and
Nub-Pex19p still interacted with Pex12-Cub-RUra3p in
pex3
cells, whereas the split-Ub measured interactions between
Pex12p and Pex15p, or Pex13p, disappear
(Fig. 7A). The absence of Pex3p
had an even more dramatic effect on Pex10-Cub-RUra3p, leaving only
its interaction with Nub-Pex19p visible on the FOA plate
(Fig. 7B). The effects of the
deletion are specific, given that the measured interaction between the
Nub- and Cub-labeled Pex11p persisted even in the
absence of Pex3p (Fig. 7E). As
was already observed for Pex12p-Cub-RUra3p in the
pex3
strain, an interaction signal between
Nub-Tom22p and Pex11-Cub-RUra3p became clearly apparent
as though Pex11p mislocalizes to the mitochondrial membrane while keeping its
oligomerization domain still intact (Fig.
7E). A tendency of some of the non-functional or only partially
functional peroxins to localize to the mitochondria was also observed in the
wild-type strain (see Fig. 4,
Tables 2,
3). Interestingly, we did not
observe an increase in proximity between the Peroxin-Cub-RUra3p and
Nub-Sec22p on deletion of PEX3
(Fig. 7A,B,E). Because
Nub-Sec22p was shown to interact with all of the ER-membrane
localized Cub-RUra3p fusion proteins tested so far
(Wittke et al., 1999
), we
conclude that the deletion of PEX3 does not cause the
membrane-associated peroxins to get trapped in the membrane of the ER.
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Discussion |
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RING-finger peroxins
The measured interactions of the three different RING-finger peroxins
Pex2p, Pex10p and Pex12p are summarized in
Fig. 8. The split-Ub assay
detected more interaction partners for Pex10p than for Pex12p and Pex2p. Among
the unique interactions of Pex10p are the measured proximities to both Pex4p
and Pex22p (Fig. 4,
Table 2). Repeating the
split-Ub assay in cells lacking PEX22 revealed the importance of
Pex22p for the stability of the Pex4p-Pex10p interaction
(Fig. 6). Together with the
measured proximity between Nub-Pex4p and
Pex22-Cub-RUra3p and two-hybrid studies by other groups (see
above), these data strongly suggest that Pex22p/Pex4p docks to the peroxin
import apparatus via a unique binding to Pex10p
(Table 2)
(Koller et al., 1999). The
RING-finger of Pex10p displays a significant sequence similarity to the RING
finger of Rad18p. Rad18p operates in the DNA repair pathway and forms a
complex with Ubc2 (Rad6p), a Pex4p-like Ub-conjugating enzyme, and the protein
Rad5p. Rad5p also contains a RING finger and physically associates with the
Ub-conjugating enzyme Ubc13p (Hoege et
al., 2002
; Ulrich and Jentsch,
2000
). Although this homology and the observed interactions are
quite suggestive, we have no direct evidence that Pex10p is an Ub-ligase and
transfers an Ub-moiety to a protein substrate
(Crane et al., 1994
;
van der Klei et al., 1998
;
Wiebel and Kunau, 1992
).
Because Pex2p was recently found in complex with other peroxins
(Reguenga et al., 2001), the
absence of any interaction partner for Pex2-Cub-RUra3p except
Nub-Pex19p is curious. There are two alternative explanations.
Pex2p exerts its function in isolation from most of the other peroxins, or the
C-terminal modification interferes with all potential binding partners or is
otherwise inept to sensing the interactions of Pex2p. The latter argument is
supported by the only partial functionality of the Pex2-Cub-RUra3p
(Table 3). In addition, we note
that a potential interaction between Pex12p and Pex2p would have escaped the
current configuration of the split-Ub assay. The Nub at the
N-terminus of Pex12p seems to point into the lumen of the peroxisome, in
accordance with Albertini et al. (Albertini
et al., 2001
), whereas the expected topology of Pex2p discouraged
us from constructing the corresponding Nub-Pex2p. The
Nub attached to the C-terminus of Pex12p did not produce any of the
interactions found for Pex12-Cub-RUra3p (Figs
2,
3). This observation confirms
our previous experience that a N-terminal attachment to Nub reduces
the sensitivity of the split-Ub assay.
Network
Although this study focuses on the interaction of the RING-finger peroxins
Pex12p, Pex10p and Pex2p, we constructed a network of interactions around
those proteins that comprised most of the other members of the peroxins as
well (Table 2,
Fig. 8). In
Fig. 8, the bars connecting the
peroxins (only in numbers) highlight the interactions between the proteins in
the split-Ub assay. The numbers accompanying the bars specify the interactions
in a certain PEX deletion strain. A plus indicates an interaction
that is dependent on the presence of a certain gene, whereas a minus indicates
an interaction that is only detected in the absence of a certain gene.
Brackets enclosing the number indicate that the interaction occurs
independently of the presence of the respective PEX gene. The
interaction of Pex10-Cub-RUra3p and Pex12-Cub-RUra3p
with Nub-Pex15p occurs only in the presence of Pex3p, whereas the
interactions of Pex19p with Pex10p, Pex12p, Pex2p and Pex11p are not affected
by a deletion of PEX3. Because the absence of Pex3p in the budding
yeast is thought to result in a lack of recognizable peroxisomal membranes
(see also Hazra et al., 2002),
we argue that the interactions between Pex19p and the other peroxins are
independent of the peroxisome and probably occur before the proteins are
integrated into the membrane. Our results therefore confirm the proposed role
of Pex19p in the recognition and insertion of peroxisomal membrane proteins
(Sacksteder et al., 2000
).
Pex15p plays an important, albeit enigmatic, role in the biogenesis of the
peroxisomes and the import of protein cargo
(Elgersma et al., 1997). The
many interactions we observe for Pex15p merely underline, yet do not explain,
this role (Figs 2,
4;
Table 2). Interestingly, Pex11p
and Pex2p do not interact with Pex15p in our assay, thereby confirming the
isolated status of both proteins in our interaction matrix
(Fig. 5). As for Pex19p, it is
difficult to imagine how Pex15p can connect to seven proteins at a time
without showing a spatial preference to any of them. It is therefore more
conceivable to assume that these interactions do not occur in a single
complex, but in either a spatially or temporally resolved manner. Pex15p as a
peroxin-specific chaperone during disassembly or readjustments of the
import-complex is one role that is compatible with the abundance of
interactions seen in our assay. Other potential roles of Pex15p, such as
serving as a scaffold protein for the members of the import complex, are also
in accordance with the observed interaction profile. However, the dependence
of the peroxin interactions on the presence of Pex15p is not general but seems
to be quite specific for the Pex4p-Pex10p interaction
(Fig. 8 and data not shown)
An interesting aspect of the interaction map concerns the increase in
proximity between Nub-Tom22p and Pex12-Cub-RUra3p and
Pex11-Cub-RUra3p on deletion of PEX3
(Fig. 7). This observation
might hint at an alternative route to the mitochondrion that is taken by some
of the peroxins in the absence of functional peroxisomes. This is not a
yeast-specific phenomenon and has been shown to occur in mammalian cell lines
lacking Pex19p (Sacksteder et al.,
2000).
We could show for the first time that the yeast Pex10p and Pex5p
multimerize in vivo (Fig. 4,
Table 2). These interactions
were already detected by biochemical means in other organisms and confirm the
general conservation of many of the peroxin interactions
(Schliebs et al., 1999).
However, some of the interactions that were reported in the literature were
not detected in our assay. An obvious explanation for missing some of those
interactions is their steric incompatibility with the N- or C-terminally
attached Nub or Cub-RUra3p. Other interactions might
have gone undetected due to an insufficient sensitivity of the assay or due to
their temporal nature. For example, the split-Ub assay does not reveal the
interactions between Pex5p and Pex13p or Pex12p
(Fig. 2,
Table 2). Because Pex5p is
thought to travel as a bearer of the cargo, some of its contacts are likely to
be very short-lived. This argument might also explain the lack of detectable
interactions between the different Cub-RUra3p modified receptors
and the PTS1-bearing Nub-fusion (Figs
2,
3,
4,
5,
6,
Table 2). In addition, some of
the missed interactions might be caused by the non- or only partial
functionality of the Nub-/Cub-constructs of the peroxins
(Table 3). Again, a prominent
example is the missed interaction between Pex13p and Pex5p. Both the
Nub- and the Cub-constructs of Pex5p are nonfunctional,
as is the Cub-construct of Pex13p. As a general rule we therefore
recommend the reader to compare the interaction data from
Table 2 with the functionality
assays in Table 3. Although a
protein that is non-functional in the peroxisomal import might still properly
intact with its binding partners, the non- or only partial functionality of a
protein makes it more likely that some of its interactions might be missed in
this assay. The presented network is therefore still rudimentary. Because this
shortcoming is shared among the different available technologies to analyze
protein complexes, a complete picture will only begin to emerge by integrating
the information from the many sources available
(Fransen et al., 2002
;
Gavin et al., 2002
;
Uetz et al., 2000
).
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Acknowledgments |
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References |
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