From the Instituto de Biologia Molecular e Celular,
Rua do Campo Alegre, 823, 4150-180 Porto, Portugal,
§ Instituto de Ciências Biomédicas Abel Salazar,
Largo do Prof. Abel Salazar, 2, 4099-003 Porto, Portugal, and
¶ Instituto de Genética Médica Jacinto
Magalhães, Praça Pedro Nunes, 88, 4050-466 Porto,
Portugal
Received for publication, November 20, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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It is now generally accepted that Pex5p, the
receptor for most peroxisomal matrix proteins, cycles between the
cytosol and the peroxisomal compartment. According to current models of
peroxisomal biogenesis, this intracellular trafficking of Pex5p is
coupled to the transport of newly synthesized peroxisomal proteins into the organelle matrix. However, direct evidence supporting this hypothesis was never provided. Here, using an in vitro
peroxisomal import system, we show that insertion of Pex5p into the
peroxisomal membrane requires the presence of cargo proteins.
Strikingly the peroxisomal docking/translocation machinery is also able
to catalyze the membrane insertion of a Pex5p truncated molecule
lacking any known cargo-binding domain. These results suggest that the
cytosol/peroxisomal cycle in which Pex5p is involved is directly or
indirectly regulated by Pex5p itself and not by the peroxisomal
docking/translocation machinery.
Peroxisomal matrix proteins are synthesized on free ribosomes and
post-translationally imported into the organelle (for review, see Refs.
1 and 2). The vast majority of proteins destined to this compartment
are recognized by Pex5p, the peroxisomal targeting signal 1 (PTS1)1 receptor (3-5).
Structurally Pex5p can be divided into two domains. The first half of
the protein has been shown to be involved in an intricate network of
protein-protein interactions (6-10). The peroxisomal targeting
information of Pex5p resides in this domain (7). The C-terminal half of
Pex5p is involved in binding the so-called PTS1 sequence, a degenerated
tripeptide present at the C terminus of practically all peroxisomal
matrix proteins (11-14).
The observation that Pex5p has a dual subcellular distribution soon led
several authors to propose that Pex5p could be a cycling receptor (5,
15, 16). According to this model, newly synthesized PTS1-containing
proteins would be recognized by cytosolic Pex5p. The cargo
protein-Pex5p complex would then dock at the peroxisomal membrane,
triggering translocation of the cargo protein across the organelle
membrane. Somewhere during this process, Pex5p would be released from
the membrane back to the cytosol to catalyze further transport cycles
(for review, see Refs. 1 and 2).
Experimental data compatible with this model were first provided by
Dodt and Gould (17); by manipulating temperature and ATP levels in
cultured mammalian cells the intracellular distribution of Pex5p could
be altered. More recently Dammai and Subramani (18) have shown that
Pex5p goes through multiple rounds of cycling between the cytosol and
the peroxisome. Thus, the two intracellular pools of Pex5p are interchangeable.
The fact that Pex5p cycles between the cytosol and the peroxisome has
been considered the landmark observation in favor of the cycling
receptor model. However, direct evidence showing that the intracellular
movement of Pex5p is coupled to the transport of PTS1-containing
proteins was never provided. Furthermore, by proposing that these two
events are coupled, the model implicitly assumes the existence of
regulatory mechanisms enabling cargo-loaded Pex5p molecules to be
inserted into the peroxisomal membrane and excluding free Pex5p
molecules from the peroxisomal docking/translocation machinery;
otherwise a futile energy-requiring cycle would be possible. This
constraint is difficult to conciliate with recent observations showing
that Pex5p molecules lacking any known cargo-binding domain are
specifically targeted to the peroxisome in vivo (7). Obviously several hypotheses can be forwarded to explain this phenomenon even in the light of the cycling receptor model. However, such a hypothesis will only be valid, and probably of utmost importance in understanding the mechanism of peroxisomal docking of Pex5p, when
definite proof for a cargo-induced peroxisomal targeting of Pex5p is provided.
Recently we described a peroxisomal in vitro import system
particularly suited to study Pex5p trafficking (19). In this work,
using the same experimental approach, we present data strongly suggesting that insertion of Pex5p into the peroxisomal membrane is
PTS1-dependent. Strikingly a truncated Pex5p molecule
lacking any known cargo-binding domain is also a substrate for the
machinery that drives insertion of Pex5p into the peroxisomal membrane. These results suggest that no crucial protein-protein interactions occur between the peroxisomal docking/translocation machinery (20-22)
on one side and the cargo proteins or the Pex5p C-terminal receptor
domain on the other. The implications of these observations on the
mechanism regulating the docking/insertion of Pex5p into the
peroxisomal membrane are discussed.
In vitro import experiments using rat liver PNS
fractions were performed in import buffer (0.25 M sucrose,
50 mM KCl, 5 mM MOPS-KOH, pH 7.2, 3 mM MgCl2, 1 mM EDTA-NaOH, pH 7.2, 0.2% (w/v) lipid-free bovine serum albumin, and 20 µM
methionine) exactly as described previously (19). The synthesis of
35S-labeled Pex5p (the large isoform) has already been
described (19). cDNAs encoding A recombinant protein containing GST fused to amino acid residues
312-639 of Pex5p (GST-TPRs) was obtained as follows. Plasmid pGEM4-Pex5p was subjected to PCR using the primers
5'-GCGAGAATTCATGGATGACCTTACGTCAGCTACCTATGA-3' and
5'-GGGTCTAGAGCGGCCGCGTCGACCTGTCACTGGGGCAGGCC-3'. The amplified fragment was inserted into the EcoRI and NotI
sites of pGEX-5X-1 plasmid (Amersham Biosciences). GST-TPRs and GST
were expressed in the XL1-Blue strain of Escherichia coli,
purified by affinity chromatography using glutathione-Sepharose 4B
(Amersham Biosciences), and dialyzed against the following solution:
0.25 M sucrose, 5 mM MOPS-KOH, pH 7.2, and 1 mM EDTA-NaOH, pH 7.2.
For the production of the fusion proteins GST-SKL and GST-LKS,
the primers 5'-GATCCCCACAATTCCCAGGTCGATCCAAGCTTTGAGC-3' and 5'-GGCCGCTCAAAGCTTGGATCGACCTGGGAATTGTGGG-3' (for GST-SKL) and 5'-GATCCCCACAATTCCCAGGTCGACTTAAGTCCTAAGC-3' and
5'-GGCCGCTTAGGACTTAAGTCGACCTGGGAATTGTGGG-3' (for GST-LKS) were
annealed. The DNA dimers were purified by non-denaturing PAGE, eluted
from the gel, and ligated to pGEX-4T-3 (Amersham Biosciences)
previously digested with the BamHI and NotI
restriction enzymes. The fusion proteins were expressed and purified as
described above. The peptides CRYHLKPLQSKL (Pep-SKL) and CRYHLKPLQLKS
(Pep-LKS) were synthesized by Sigma Genosys.
Insertion of Pex5p into the Peroxisomal Membrane Is
PTS1-dependent--
In a recent work, we described a
cell-free in vitro import system to study Pex5p association
with and release from the peroxisomal compartment (19). In this work,
we have used this experimental system to address a crucial issue in the
field of peroxisomal biogenesis: is the intracellular cycling of Pex5p
coupled to the transport of PTS1-containing proteins across the
peroxisomal membrane?
As an attempt to solve this question, we first tried to determine
whether or not import of Pex5p into the peroxisome could be stimulated
by supplementing the import reaction with a PTS1-containing recombinant
protein, GST-SKL. The concentration of GST-SKL in the import assays was
8 µM, a value more than 100-fold the dissociation constant reported for the Pex5p-PTS1 complex (14). Considering that
Pex5p is a low abundance protein in rat liver (0.008% of total liver
protein; calculated from the data in Ref. 20), this concentration of
GST-SKL should ensure an almost complete saturation of Pex5p. As a
negative control, we used GST-LKS, a glutathione S-transferase molecule containing at its C terminus a
non-functional PTS1-like sequence (23). The results of this experiment
are shown in Fig. 1A; no
stimulation on the import of in vitro synthesized Pex5p
could be observed.
We repeated this experiment but this time using a PTS1-containing
peptide (Pep-SKL). The biological activity of this peptide is well
documented (23, 24). Although the concentration of the peptide used in
this experiment was 350-fold the dissociation constant of the
Pex5p-PTS1 complex, again no stimulation on the peroxisomal import of
Pex5p could be detected (see Fig. 1B).
There are two possibilities to explain this negative result: either
insertion of Pex5p into the peroxisomal membrane is not coupled to the
transport of cargo proteins or the cytosolic phase of the PNS fractions
used in these import assays already has a sufficient amount of
PTS1-containing proteins. Considering that 1) rat liver peroxisomes are
particularly prone to disruption during tissue homogenization and
represent 2% of total liver protein (25) and 2) peroxisomal proteins
are not processed after import, i.e. they retain their
peroxisomal targeting information (26), we reasoned that the second
possibility should be tested.
If insertion of Pex5p into the peroxisomal membrane occurs only when
the PTS1 receptor is in a complex with cargo proteins, then
sequestering these cargo proteins should inhibit the peroxisomal import
of 35S-labeled Pex5p. For this purpose a GST recombinant
protein containing amino acid residues 312-639 of Pex5p (GST-TPRs) was
produced. This domain of Pex5p contains the PTS1 binding activity of
Pex5p but lacks its peroxisomal targeting information (7, 10).
As shown in Fig. 2, when in
vitro synthesized Pex5p is subjected to an import reaction in the
presence of 0.17 µM GST-TPRs, no protease-resistant Pex5p
can be detected (Fig. 2, A and B, lanes
6). Adding the same amount of GST to the import reaction has no
effect on the import of Pex5p into the organelle (Fig. 2, lanes
3; see also Fig. 3B).
When these import assays are performed in the presence of a GST fusion
protein (GST-LKS) or a peptide (Pep-LKS), both containing a
non-functional PTS1-like sequence, the inhibitory properties of
GST-TPRs on the in vitro import of Pex5p remain unchanged
(Fig. 2, lanes 5). In sharp contrast, when GST-SKL or
Pep-SKL are used under the same conditions this inhibition is partially
reverted (Fig. 2, lanes 4; see legend to Fig. 2). The reason
why a complete reversion is not observed is not known at the moment. It
is possible that addition of GST-TPRs to PNS fractions not only
titrates PTS1-containing proteins but also some other(s) component(s)
necessary for the efficient targeting of Pex5p to the peroxisomal
compartment. Although additional work will be necessary to clarify this
matter the data presented here are clear in one point: insertion of
Pex5p into the peroxisomal membrane is PTS1-dependent.
C-terminal Truncated Versions of Pex5p Are Substrates for the
Peroxisomal Docking/Insertion Machinery--
Recently Dodt et
al. (7) have mapped the region of Pex5p responsible for its
peroxisomal targeting. After transfection of human fibroblasts with
plasmids encoding epitope-tagged N-terminal fragments of Pex5p the
authors were able to show a peroxisomal location for a recombinant
protein containing just the first 214 amino acid residues of Pex5p.
Although the exact peroxisomal location of this recombinant protein was
not defined in that study (i.e. no distinction between Pex5p
molecules inserted into the peroxisomal membrane or just adsorbed at
the surface of the organelle was made), the fact that this domain of
Pex5p lacks any cargo protein-interacting domain and yet is correctly
targeted to the peroxisome led us to investigate this phenomenon in
more detail.
Thus, we tried to determine whether or not C-terminal truncated
versions of Pex5p still retain the capacity of being inserted into the
peroxisomal membrane. Two different 35S-labeled proteins
were synthesized:
The results of this experiment are shown in Fig. 3B.
GST-TPRs has no effect on the peroxisomal import of both
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
C1Pex5p and
C2Pex5p preceded
by the T7 RNA polymerase promotor were obtained by PCR amplification of pGEM4-Pex5 (19) using the forward primer
5'-AGTCAGTGAGCGAGGAAGCGGAAGAGC-3' and the reverse primers
5'-CGCGCTCTTTCATTAGTACCCCTTATCATA-3' (for
C1Pex5p) or
5'-GTCCCGCTATTACGTGTGCTGCAGATCCTC-3' (for
C2Pex5p). These
cDNA fragments were then subjected to in vitro
transcription/translation as described previously (19).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (48K):
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Fig. 1.
The basal level of peroxisomal in
vitro import of Pex5p is not increased by PTS1-containing
peptides/proteins. A, PNS fractions were incubated with
35S-labeled Pex5p in import buffer containing 5 mM ATP in the presence of 8 µM GST
(lane GST), GST-LKS (lane LKS), or GST-SKL
(lane SKL) fusion proteins for 30 min at 26 °C. Samples
were then treated with proteinase K to degrade non-imported
35S-labeled Pex5p, and the organelles were pelleted and
subjected to SDS-PAGE. An autoradiograph of the gel is shown.
B, PNS fractions were incubated with 35S-labeled
Pex5p in import buffer containing 5 mM ATP either in the
absence (lane ) or in the presence of synthetic peptides
(25 µM final concentration) CRYHLKPLQSKL (lane
SKL) and CRYHLKPLQLKS (lane LKS). Samples were
processed as described above. Stage 2, a peroxisomal
population of Pex5p partially accessible to proteinase K (19).
Lane L, 35S-labeled Pex5p (10% of the input in
each import reaction).
View larger version (44K):
[in a new window]
Fig. 2.
Insertion of Pex5p into the peroxisomal
membrane is PTS1-dependent. In vitro
synthesized Pex5p was incubated with PNS fractions in the presence of
0.17 µM GST (lanes 1-3) or GST-TPRs
(lanes 4-6). In A, GST-SKL (lanes 1 and 4) or GST-LKS (lanes 2 and 5)
fusion proteins were also included in the import reactions (8 µM final concentration). In B, import
reactions received Pep-SKL (lanes 1 and 4),
Pep-LKS (lanes 2 and 5), or no peptide
(lanes 3 and 6). Peptides were used at 25 µM final concentration. All import reactions were
performed in the presence of 5 mM ATP. After 30 min at
26 °C, protein samples were processed as described in the legend to
Fig. 1. The ratio of stage 2 Pex5p in lanes 4 to lanes
1 is 0.64 ± 0.11 (n = 4) and 0.31 ± 0.08 (n = 4) in A and B,
respectively. Stage 2 and Lane L, see the legend
to Fig. 1.
View larger version (32K):
[in a new window]
Fig. 3.
C-terminal truncated Pex5p molecules are
substrates for the peroxisomal docking/insertion machinery.
A, in vitro synthesized C1Pex5p and
C2Pex5p
were subjected to import reactions in the presence (lanes +)
or absence (lanes
) of exogenous ATP. After 30 min at
26 °C, protein samples were processed as described in the legend to
Fig. 1. Stage 3, a peroxisomal population of Pex5p
completely resistant to the action of proteinase K (19); Stage
2, see the legend to Fig. 1. Lanes L,
35S-labeled
C1Pex5p or
C2Pex5p (1% of the input in
each import reaction). The numbers at the left
indicate the molecular masses of the applied standards in kDa.
B, insertion of
C1Pex5p and
C2Pex5p into the
peroxisomal membrane is not inhibited by GST-TPRs.
35S-Labeled full-length Pex5p (FL) or the
truncated versions
C1Pex5p (
C1) or
C2Pex5p
(
C2) were incubated with PNS fractions in the absence
(lanes
) or in the presence of GST (lane GST)
or GST-TPRs (lane TPRs) fusion proteins. Import reactions
were performed in the presence of 5 mM ATP. After
proteinase K treatment, the organelles were isolated and subjected to
SDS-PAGE. Lanes L, reticulocyte lysates containing
35S-labeled full-length Pex5p (panel FL) or the
C1Pex5p and
C2Pex5p truncated versions (panels
C1 and
C2, respectively).
C1Pex5p, amino acid residues 1-324 of Pex5p, and
C2Pex5p, amino acid residues 1-197 of Pex5p. Import reactions were
performed in the presence or absence of exogenous ATP as described
previously (19). The results of this experiment are shown in Fig.
3A. In both cases, protease-resistant species corresponding
to stage 2 Pex5p (lanes + ATP) and stage 2 plus stage 3 Pex5p (lanes
ATP) can be easily detected (see legends to Figs. 1 and 3 and Ref. 19). This result could suggest that
amino acid residues 1-197 of Pex5p are sufficient to drive insertion
of Pex5p into the peroxisomal membrane. There is, however, another
possibility. In our experimental system import of these truncated forms
of Pex5p is performed in the presence of endogenous rat liver Pex5p.
Recently it was suggested that Pex5p may form a homodimer or a
homotetramer and that this polymeric Pex5p is probably the active form
of the PTS1 receptor (27, 28). Thus, in principle, the
35S-labeled proteins used here could interact with
endogenous Pex5p, which in turn would drive insertion of
C1Pex5p and
C2Pex5p into the peroxisomal membrane. One easily testable
prediction of such a hypothesis is that if import of full-length Pex5p
is inhibited (e.g. by exploring the properties of GST-TPRs),
then import of
C1Pex5p and
C2Pex5p should also be inhibited.
C1Pex5p and
C2Pex5p. Thus, our data confirm and extend the observations made by
Dodt et al. (7): the N-terminal domain of Pex5p can be
targeted to and inserted into the peroxisomal membrane. This
observation may have major implications on the mechanism regulating the
process of docking/insertion of Pex5p into the peroxisomal membrane.
Indeed, the fact that
C2Pex5p is a substrate for the
docking/insertion machinery suggests that no crucial protein-protein
interactions occur between this machinery on one side and the receptor
domain of Pex5p or the cargo proteins on the other. If this proves to be the case, then how is cycling of Pex5p regulated? We can think of
only one possibility: Pex5p itself directly or indirectly regulates this cycle. Many different hypotheses can be envisaged to explain such
a mechanism. For instance, binding of cargo proteins to the receptor
domain of Pex5p could induce conformational alterations on the PTS1
receptor, activating (e.g. exposing) its peroxisomal targeting domain; this regulatory mechanism would have been lost in
both
C1Pex5p and
C2Pex5p (i.e. the peroxisomal
targeting domain in these truncated molecules would be constitutively
active). Data suggesting that Pex5p N-terminal and C-terminal domains
interact with each other are already available and could support this
hypothesis (27). Alternatively cytosolic Pex5p is kept away from the
peroxisomal compartment due to an interaction with some (still unknown)
protein. Such a putative factor would only bind to cargo-unloaded Pex5p molecules through an interaction requiring the receptor domain of Pex5p
(which is not present in both
C1Pex5p and
C2Pex5p).
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FOOTNOTES |
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* This work was supported by Grants PRAXIS XXI/BD/20043/99 (to A. M. G.), SFRH/BD/1445/2000 (to C. P. G.), PRAXIS XXI/BD/21819/99 (to M. E. O.), and POCTI/BME/34648/99 from Fundação para a Ciência e Tecnologia, Portugal.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Instituto de
Biologia Molecular e Celular, Rua do Campo Alegre, 823, 4150-180 Porto,
Portugal. Tel.: 351-226074900; Fax: 351-226099157; E-mail: jazevedo@ ibmc.up.pt.
Published, JBC Papers in Press, December 26, 2002, DOI 10.1074/jbc.C200650200
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ABBREVIATIONS |
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The abbreviations used are: PTS, peroxisomal targeting sequence; GST, glutathione S-transferase; PNS, rat liver postnuclear supernatant; TPRs, Pex5p domain (amino acid residues 312-639) comprising its tetratricopeptide repeats; MOPS, 4-morpholinepropanesulfonic acid.
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