From the Instituto de Biologia Molecular e Celular,
Rua do Campo Alegre, 823, 4150-180 Porto, Portugal, the
§ Instituto de Ciências Biomédicas Abel Salazar,
Universidade do Porto, 4099-003 Porto, Portugal, and the
¶ Instituto de Genética Médica Jacinto
Magalhães, 4050-466 Porto, Portugal
Received for publication, September 16, 2002, and in revised form, October 21, 2002
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ABSTRACT |
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According to current models of peroxisomal
biogenesis, Pex5p cycles between the cytosol and the peroxisome
transporting newly synthesized proteins to the organelle matrix.
However, little is known regarding the mechanism of this pathway. Here,
we show that Pex5p enters and exits the peroxisomal compartment in a
process that requires ATP. Insertion of Pex5p into the peroxisomal
membrane is blocked by anti-Pex14p IgGs. At the peroxisomal level, two Pex14p-associated populations of Pex5p could be resolved, stage 2 and
stage 3 Pex5p, both exposing the majority of their masses into
the organelle lumen. Stage 3 Pex5p can be easily
detected only under ATP-limiting conditions; in the
presence of ATP it leaves the peroxisomal compartment
rapidly. Our data suggest that translocation of PTS1-containing
proteins across the peroxisomal membrane occurs concomitantly with
formation of the Pex5p-Pex14p membrane complex and that this is
probably the site from which Pex5p leaves the peroxisomal compartment.
The vast majority of peroxisomal matrix proteins is targeted to
the organelle via the so-called peroxisomal targeting signal 1 (PTS1)1 pathway (reviewed in
Refs. 1 and 2). Proteins belonging to this family contain at their
extreme C terminus the sequence Ser-Lys-Leu (or a variant
(3-5)) that is recognized by Pex5p, the PTS1 receptor (6-8). The
observation that Pex5p is predominantly found in the cytosol with only
a small fraction associated with the peroxisome led several authors to
propose the so-called cycling receptor mechanism (8-10). According to
this model, newly synthesized PTS1-containing peroxisomal proteins
would be recognized by cytosolic Pex5p, transported to the surface of
the peroxisome, and translocated across the membrane of the organelle.
Experimental evidence compatible with this model was first provided by
Dodt and Gould (11), reversible accumulation of Pex5p at the
peroxisomal compartment could be demonstrated in vivo by
manipulating ATP levels and temperature in cultured human fibroblasts.
More recently, Dammai and Subramani (12) were able to show that Pex5p
goes through multiple rounds of cycling between the cytosol and the peroxisome.
Peroxisomal membrane docking, translocation of cargo proteins, and
recycling of Pex5p requires a complex machinery. In the last years,
more than 20 peroxins have been identified in several organisms, most
of them involved in the Pex5p-mediated peroxisomal protein import
(reviewed in Refs. 1 and 2). However, our knowledge on the mechanism by
which peroxisomal matrix proteins are translocated across the organelle
membrane is still very limited. Much of what is known regarding this
matter derives either from steady-state level analysis of peroxisomal
Pex5p on several mutant cell lines (e.g. Ref. 13) or from
studies aiming at the characterization of peroxin-peroxin interactions
(see Ref. 14 and references cited therein). Collectively, these data
provide the basis for a model in which peroxins are classified into
three functional groups: 1) docking proteins; 2) proteins involved in
the translocation of matrix proteins across the peroxisomal membrane;
and 3) proteins mediating the release (recycling) of Pex5p from the
peroxisomal compartment (reviewed in Refs. 1 and 2). Although many
aspects of this model may indeed be correct, the recent isolation of
stable protein complexes comprising peroxins belonging to different
functional groups raises some questions regarding this classification
(15, 16). Here, we provide data suggesting that insertion of Pex5p into
the peroxisomal membrane requires Pex14p, a proposed docking protein
(17-19). However, Pex14p-associated Pex5p exposes the majority of its
mass into the peroxisomal matrix suggesting that translocation of cargo
proteins into the matrix of the organelle occurs concomitantly with the
formation of the Pex5p-Pex14p membrane complex. Furthermore, Pex14p-associated Pex5p is exported from the peroxisomal compartment in
an ATP-dependent process. Thus, our results suggest, on one hand, that translocation of peroxisomal matrix proteins across the
organelle membrane occurs through a Pex14p-containing structure via
Pex5p, raising the possibility that no other peroxins other than Pex5p
interact with the PTS1-containing proteins during the membrane
translocation event and, on the other hand, that Pex5p is retained at
the peroxisomal membrane during all steps of this process.
Synthesis of Radiolabeled Proteins--
The cDNA encoding
full-length human Pex5p (the large isoform (20, 21) cloned in the
pGEMTM-T Easy vector (pGEMT-Pex5) (22) was inserted into
the SalI site of pGEM-4 (Promega) originating pGEM4-Pex5.
This plasmid was transcribed in vitro using T7 RNA
polymerase (Roche Molecular Biochemicals). 35S-Labeled
Pex5p was synthesized using the translation kit Reticulocyte Type II
(Roche Molecular Biochemicals) in the presence of
[35S]methionine (specific activity >1000 Ci/mmol; ICN
Biomedicals, Inc.) following the standard conditions of the manufacturer.
Peroxisomal in Vitro Import Experiments--
Post-nuclear
supernatants (PNS) from rat liver were prepared from male Wistar rats
(1-2 months of age). Livers were rapidly removed and homogenized in
SEM buffer (0.25 M sucrose, 5 mM MOPS-KOH, pH
7.2, and 1 mM EDTA-NaOH, pH 7.2). PNS fractions were
obtained by centrifuging the homogenate two times at 600 × g for 10 min at 4 °C. Aliquots of the PNS fraction (at
40-60 mg/ml) were frozen in liquid nitrogen and stored at
Import reactions (100 µl final volume) 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) using 150 µg of rat
liver PNS protein and 0.5 µl of the reticulocyte lysate containing
35S-labeled Pex5p. In some experiments the concentration of
lipid-free bovine serum albumin in the import buffer was decreased to
0.002% (w/v) (import buffer B). Where indicated, nucleotides (100 mM stock solutions, pH 7.2, with NaOH; Sigma) were used at
1 mM final concentration. The contributions of the
reticulocyte lysate and rat liver PNS fraction to the levels of ATP in
import reactions were not determined experimentally. Nevertheless,
considering the total adenine nucleotide pool present in the
reticulocyte lysate (Roche Molecular Biochemicals) and rat liver (23),
this value was estimated to be lower than 36 µM. At the
end of the import reaction, the samples were treated with proteinase K
(300 µg/ml final concentration) for 30 min at 4 °C. The protease
was inhibited with 500 µg/ml phenylmethysulfonyl fluoride
(added from a 50 mg/ml stock solution in ethanol) for 2 min on ice. The
import reaction was diluted to 1 ml with SEM buffer and the organelles were isolated by centrifugation at 15,000 × g for 15 min at 4 °C. The samples were subjected to SDS-PAGE, transferred to
nitrocellulose, and the radioactive proteins were detected by
autoradiography. Under these conditions optimum import of Pex5p is
observed at 26 °C (tested temperatures: 0, 8, 16, 26, and 37 °C)
and occurs linearly during the first 45 min of incubation (data not shown).
In antibody inhibition experiments, 150 µg of PNS protein, in 30 µl
of import buffer, were preincubated with 20 µg of purified immunoglobins (5 µg/µl in PBS) for 20 min on ice before starting the import reaction. GST-Pex5p and GST recombinant proteins were used in import reactions at a final concentration of 100 µg/ml.
Immunoprecipitation of in Vitro Imported Pex5p--
Two aliquots
of a PNS fraction (1.3 mg of protein) were incubated in import buffer
either in the presence or absence (mock-treated sample) of 5 µl of a
reticulocyte lysate containing 35S-labeled Pex5p. At the
end of the incubation (30 min at 26 °C) both samples were treated
with proteinase K as described above. The protease was inhibited and
the organelles were diluted with 1 ml of SEM buffer containing 50 µg/ml phenylmethysulfonyl fluoride and 1:100 (v/v) mammalian protease
inhibitors mixture (Sigma). After centrifugation (15,000 × g for 15 min at 4 °C) to sediment the organelles,
35S-labeled Pex5p (0.1 µl) was then added to the
mock-treated sample. To avoid nonimport related Pex5p-Pex14p
interactions, 10 µg of GST-Pex5p was added to each tube. Both samples
were then solubilized in 1.3 ml of immunoprecipitation buffer (1%
(w/v) Nonidet P-40, 100 mM potassium acetate-acetic acid,
pH 7.4, 2 mM EDTA-NaOH, pH 8.0, 100 µg/ml
phenylmethysulfonyl fluoride, and 1:100 (v/v) mammalian protease
inhibitors mixture) for 15 min on ice and subjected to centrifugation
(60,000 × g for 15 min at 4 °C) to remove insoluble material. One aliquot (100 µl) of each supernatant was kept on ice
during the rest of the procedure and subjected to trichloroacetic acid
precipitation (10% (w/v) final concentration) before SDS-PAGE analysis. The remaining supernatants were divided into two halves. One-half received 5 mg of protein A-Sepharose beads previously incubated with 5 µl of anti-Pex14p serum; the other half received the
same amount of beads previously incubated with 5 µl of preimmune serum. These samples were incubated for 60 min at 4 °C with gentle agitation, the beads were sedimented by centrifugation and washed three
times with 1 ml of immunoprecipitation buffer. Immunoprecipitated proteins were eluted with 60 µl of Laemmli sample buffer and
subjected to SDS-PAGE (24).
Miscellaneous--
Centrifugation of PNS fractions on step
Nycodenz gradients (1.5 ml of 45% (w/v), 6.5 ml of 30% (w/v),
and 1 ml of 25% (w/v) Nycodenz in 5 mM MOPS-KOH, pH 7.2, and 1 mM EDTA-NaOH, pH 7.2) was performed in an angular
rotor (65.13, Sorvall) at 60,000 × g for 45 min at
4 °C. Fractionation of the gradients, determination of the
activities of marker enzymes, and trichloroacetic acid precipitation of
gradient fractions were performed exactly as described (25).
Antibodies directed to human Pex14p (16) and Pex5p (22) were described
before. Antibodies directed to rat PMP70 (Zymed Laboratories, Inc. (9,
26)), catalase (Research Diagnostics, Inc.), and GST (Amersham
Biosciences) were purchased. Rabbit and goat antibodies were detected
on Western blots using alkaline phosphatase-conjugated anti-rabbit or
anti-goat antibodies, respectively (Sigma). IgGs were purified from
rabbit sera using protein A-Sepharose beads according to the
manufacturer (Amersham Biosciences). Preparation of Fab fragments using
immobilized papain was carried out using the ImmunoPure Fab Preparation
Kit (Pierce).
For expression of the fusion protein GST-PEX5p, the plasmid pGEMT-Pex5
was used as template in a PCR reaction using the upper primer
5'-GGCGTCGACTGGAGATCTATGGCAATGCGGGAGCTGGTGGA-3' and the lower
primer 5'-GGGTCTAGAGCGGCCGCGTCGACCTGTCACTGGGGCAGGCC-3'. The
amplified DNA fragment was digested with the enzymes BglII and NotI and cloned into the BamHI and
NotI sites of pGEX-4T-3 (Amersham Biosciences). GST-Pex5p
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
SEM buffer.
Digestion of proteins with Genenase I (2 units/digestion; New
England BioLabs) was performed in a buffer containing 0.25 M sucrose, 20 mM Tris-HCl, pH 8.0, 1 mM EDTA-NaOH, pH 8.0, 1 mM dithiothreitol, 100 mM NaCl, 50 µg/ml phenylmethysulfonyl fluoride, and 1%
(w/v) Triton X-100 for 30 min at 23 °C. Digestions were stopped with
trichloroacetic acid (10% (w/v) final concentration) and the samples
were subjected to SDS-PAGE. Protease protection assays were performed
as described (22).
In Vitro Synthesized Pex5p Is Specifically Imported into
Peroxisomes Using a Cell-free System--
Transport of PTS1-containing
proteins across the peroxisomal membrane requires the concerted action
of many different peroxins. Although a significant fraction of this
machinery resides and functions in the peroxisomal compartment, there
are several peroxins (Pex5p included) that seem to exist in a dynamic
equilibrium between the cytosol and the peroxisome. Furthermore,
presently, the involvement of other subcellular compartments in the
process of peroxisomal biogenesis cannot be formally excluded (reviewed
in Ref. 27). Thus, we just decided to use a PNS to set up a
peroxisomal in vitro import system.
To test the validity of this strategy, in vitro synthesized
35S-labeled Pex5p (the large isoform (20, 21)) was
incubated with a rat liver PNS in import buffer (see "Experimental
Procedures") containing 1 mM ATP, 1 mM
ATP
To provide evidence regarding the peroxisomal location of in
vitro imported Pex5p, import experiments in the presence or
absence of exogenous ATP were performed. After import, one-half of each import reaction was treated with proteinase K; the other halves received buffer only. The samples were then loaded onto the top of
discontinuous Nycodenz gradients and centrifuged. The gradients were
fractionated, and the distributions of in vitro synthesized Pex5p, endogenous Pex14p, and several marker enzymes were determined. As shown in Fig. 1B (panels A and B),
both catalase and endogenous Pex14p display a dual behavior under these
conditions: one fraction is recovered in fractions 2 and 3 and
corresponds to highly pure peroxisomes; the other fraction remains at
the top of the gradient and probably reflects the existence of
peroxisomes of lower density (28) together with some
disrupted organelles. When the behavior of 35S-labeled
Pex5p in the samples not treated with protease is analyzed, a quite
asymmetric distribution is observed (Fig. 1B, panel
C; shown only for the sample subjected to import in the presence of exogenous ATP). Most of the radiolabeled protein remains at the top
of the gradient and only a small fraction is found in fractions 2 and
3. The majority (but not all) of the 35S-labeled Pex5p
staying at the top of gradient represents nonimported peroxin, as
expected. Indeed, when the behaviors of stage 2 Pex5p (panel
D) and stage 2 plus stage 3 Pex5p (panel E) are
analyzed a more symmetric distribution, paralleling the one displayed
by endogenous Pex14p, is observed. Clearly, both peroxisomal
populations detected in these kinds of gradients are import-competent
for Pex5p. Taken together, these data indicate that in vitro
synthesized Pex5p can be specifically imported into peroxisomes.
Characterization of the Pex5p Membrane Insertion
Step--
Previously, we provided data suggesting that endogenous
Pex5p from rat liver peroxisomes is a subunit of a Pex14p-containing complex (16, 22). On the other hand, several authors have proposed that
Pex14p is the docking protein for Pex5p (17-19). Thus, the possible
involvement of Pex14p on the in vitro import of Pex5p was
assessed on both functional and structural levels.
We first asked whether antibodies directed to Pex14p block in
vitro import of Pex5p. For this purpose, rat liver PNS fractions were preincubated with IgGs directed to either Pex14p or PMP70 (the
most abundant peroxisomal membrane protein (26)) and subjected to an
import reaction. The relative amounts of Pex5p in stage 2 and stage 3 in the two samples were compared. As shown in Fig. 2A, IgGs directed to Pex14p
strongly inhibit the appearance of protease-protected Pex5p. Virtually
the same inhibition was observed when the import reaction was performed
in the presence of ATP (data not shown). These experiments were also
performed using Fab fragments directed to Pex14p. Although these Fab
fragments were as efficient as the precursor IgGs in recognizing Pex14p in Western blots, we were unable to see an inhibitory effect of these
fragments on the in vitro import rate of Pex5p (data not shown). It is likely that the observed import inhibition with anti-Pex14p IgGs results from sterical hindrance effects and not by
blocking of the Pex5p-binding site on Pex14p. The Pex5p-binding domain
of Pex14p (comprising the first 78 amino acid residues of Pex14p (29))
is probably not exposed into the cytosol
(30).2 Thus, taken together,
these data do not prove that Pex14p is the initial peroxisomal docking
protein for Pex5p, but they do indicate that Pex14p and/or some
Pex14p-bound component(s) is functionally involved in the formation of
stage 2 and/or stage 3 Pex5p.
Data demonstrating a structural involvement of Pex14p on the import
pathway of Pex5p was obtained as follows. 35S-Labeled Pex5p
was subjected to an import reaction in the absence of exogenous ATP.
After proteinase K treatment, the organelles were solubilized using a
mild detergent and subjected to immunoprecipitation using the
anti-Pex14p antibody. As shown in Fig. 2B, both stage 2 and
stage 3 Pex5p can be co-immunoprecipitated with the anti-Pex14p antibody. Control experiments (see legend to Fig. 2) clearly indicate that the observed immunoprecipitation is specific and occurs only when
35S-labeled Pex5p was subjected to an import reaction.
Based on these data, we conclude that both stage 2 and stage 3 Pex5p
are components of a Pex14p-containing complex.
Relationship between Stage 2 and Stage 3 Pex5p--
As shown
above, when 35S-labeled Pex5p is subjected to an import
reaction in the absence of exogenous ATP, two different
protease-resistant populations (stage 2 and stage 3 Pex5p) are
obtained. When the import kinetics of these two species are analyzed
(see Fig. 3), no clear relationship
between stage 2 and stage 3 Pex5p can be established, although it seems
that stage 2 Pex5p can be detected earlier than stage 3 Pex5p.
To clarify this point, we tried to explore the fact that recombinant
GST-Pex5p inhibits the import of in vitro synthesized Pex5p
into peroxisomes (see Fig.
4A). Furthermore, in the
absence of exogenous ATP, GST-Pex5p is unable to displace stage 2 and stage 3 Pex5p from the peroxisomal compartment (Fig. 4B). In
the presence of ATP a different result is obtained. After addition of
the recombinant protein to the import reaction, the amount of stage 2 Pex5p decreases slowly over time (Fig. 4B). There are two
possibilities to explain this result: either GST-Pex5p is displacing
35S-labeled Pex5p from the peroxisomal compartment in an
ATP-dependent process; or, more likely, in vitro
synthesized Pex5p simply exits the peroxisome under these conditions
(as observed for the endogenous peroxin, see below), in a process that
is not directly related to the presence of GST-Pex5p but that can only
be detected when import de novo is blocked. Independently of
the reasons behind this event, we reasoned that the properties
displayed by GST-Pex5p could still be explored to establish a
relationship between stage 2 and stage 3 Pex5p.
For this purpose, two in vitro pulse-chase import
experiments were performed. In the first experiment, Pex5p was imported into peroxisomes for 20 min in the presence of ATP. GST-Pex5p was then
added to stop de novo import of Pex5p and, 7 min later, ATP Export of Endogenous Rat Liver Pex5p from the Peroxisome Requires
ATP Hydrolysis--
To obtain independent evidence supporting the
existence and properties of stage 2 and stage 3 Pex5p, we have repeated
some of the experiments described above but this time the behavior of
the endogenous (rat liver) Pex5p was monitored. PNS fractions were
incubated in import buffer under several conditions (see Fig.
6). The samples were then treated with
proteinase K, the organelles were isolated by centrifugation and
analyzed by Western blotting. As shown in Fig. 6 (panel A),
both stage 2 and stage 3 Pex5p can be detected in organelles incubated
at 4 °C in the absence of exogenous ATP (lane 1).
Interestingly, the ratio of endogenous stage 2 to stage 3 Pex5p is much
smaller than observed in the in vitro import experiments
performed in the absence of exogenous ATP, suggesting that the majority
of peroxisome-associated rat liver Pex5p is just on its way to the
cytosol. Similar ratios of stage 2 to stage 3 Pex5p can be observed
when PNS fractions are incubated in the presence of 1 mM
ATP
In contrast with the results obtained with the in vitro
imported Pex5p, the amount of endogenous stage 2 Pex5p does not
increase in the presence of ATP at 26 °C. It should be noted that
only 1-5% of the in vitro synthesized peroxin used in our
import assays is imported into peroxisomes. Assuming similar values for
the endogenous peroxin and considering that in rat liver the ratio of
cytosolic Pex5p to peroxisomal Pex5p is only 6:1 (22) a low level of
import would be expected.
Membrane Topology of Stage 2 and Stage 3 Pex5p--
In a previous
work (22), it was shown that Pex5p from purified rat liver peroxisomes
is quite resistant to exogenous proteases as long as the peroxisomal
compartment is left intact (22). When this is not the case
(e.g. if Triton X-100 is used in the protease assays) Pex5p
is completely degraded by the proteases. These observations were based
on Western blot analysis using a polyclonal antibody
directed to Pex5p. Because the Pex5p epitopes recognized by this
antibody are not known, no conclusions regarding the extent of exposure
of Pex5p to the peroxisomal matrix could be withdrawn from those
experiments. In this work, the availability of radiolabeled peroxisomal
Pex5p prompted us to repeat those protease assays. Thus, an in
vitro import experiment in the absence of exogenous ATP was
performed to produce stage 2 and stage 3 Pex5p. The organelles were
isolated by centrifugation, resuspended gently, and incubated with
proteinase K under several conditions. As shown in Fig.
7, proteinase K treatment of intact
organelles resulted in two bands (stage 2 and stage 3 Pex5p), as
expected. However, when detergent-solubilized or sonicated organelles
are subjected to the same treatment only small radiolabeled peptides can be detected. These results strongly suggest that the bulky part of
Pex5p is exposed into the peroxisomal matrix.
In an attempt to define more precisely the membrane topology of stage 2 Pex5p, we tried to determine which terminus of this protein was exposed
into the cytosol. This was accomplished by using Genenase I. This engineered protease cleaves only once to GST-Pex5p (Fig.
8A). Analysis of the cleavage
products obtained with GST-Pex5p clearly indicate that Genenase I
cleaves at the N terminus of Pex5p. Thus, we used this protease to map
the Pex5p domain that is cleaved when intact organelles are subjected
to proteinase K treatment. As shown in Fig. 8B, both
in vitro imported stage 2 Pex5p and Pex5p from purified rat
liver peroxisomes are no longer cleaved by Genenase I after proteinase
K treatment. We conclude that the N terminus (and not the C-terminal
receptor domain) of stage 2 Pex5p is exposed into the cytosol.
In this work, a characterization of Pex5p entry into and exit from
the peroxisomal compartment is presented. Using a postnuclear supernatant from rat liver, specific import of in vitro
synthesized Pex5p into peroxisomes was demonstrated by three
independent lines of evidence: first, in vitro imported
Pex5p acquires the membrane topology of the endogenous peroxin; second,
Nycodenz gradient centrifugation of PNS fractions subjected to import
reactions show a typical peroxisomal distribution for the in
vitro synthesized peroxin; finally, insertion of Pex5p into a
protease-resistant location is strongly inhibited by anti-Pex14p IgGs.
Import of Pex5p in the absence of exogenous ATP revealed the existence
of two different protease-resistant populations. The existence of these
two populations was also confirmed with the endogenous (rat liver)
Pex5p. The first population (stage 2 Pex5p) exposes ~2 kDa of its N
terminus into the cytosol. The second population (stage 3 Pex5p) is
completely inaccessible to proteinase K. Thus, stage 3 Pex5p could
represent an intraperoxisomal population of the PTS1 receptor, a
hypothesis that would be in agreement with the extended version of the
cycling receptor model (12). However, it should be noted that stage 3 Pex5p can be co-immunoprecipitated with Pex14p, a peroxisomal membrane
protein (30). Furthermore, neither stage 2 nor stage 3 Pex5p can be
extracted from the peroxisomal membrane by sonication in the presence
of low or high ionic strength buffers.2 Thus, stage 3 Pex5p
is still a membrane-associated protein. Finally, both stage 2 and stage
3 Pex5p can be completely degraded by proteinase K when the peroxisomal
membrane is disrupted prior to protease treatment. Taken together,
these data indicate that the bulky part of these two Pex5p populations
(the tetratricopeptide repeat-containing domain included) is
exposed into the matrix of the peroxisome. Considering that import of
Pex5p into peroxisomes is strongly inhibited by anti-Pex14p IgGs and
that both stage 2 and stage 3 Pex5p can be immunoprecipitated with
anti-Pex14p antibodies, our Pex5p topology results have a major
implication: translocation of newly synthesized peroxisomal proteins
across the peroxisomal membrane probably occurs concomitantly with the
formation of the Pex5p-Pex14p complex. Thus, Pex14p is not just part of
the docking site for cargo-loaded Pex5p but is, most likely, a member
of the translocation machinery itself. Recent data from our and other laboratories corroborate this interpretation: using mild solubilization conditions both mammalian (16) and yeast Pex14p (15) can be isolated in
a complex containing several peroxins presumably involved in the
translocation step of peroxisomal proteins across the organelle membrane.
Using in vitro import pulse-chase experiments we provide
data suggesting that stage 2 is the precursor of stage 3 Pex5p and that
stage 3 Pex5p leaves the peroxisomal compartment rapidly in the
presence of ATP. An observation compatible with these data was obtained
when the behavior of endogenous rat liver Pex5p was monitored: ATP (but
not ATP The fact that stage 3 Pex5p exits the peroxisomal compartment in a
ATP-dependent process, together with the observation that this Pex5p population can be immunoprecipitated by an anti-Pex14p antibody provide the basis for the second major conclusion of this
work: Pex14p-associated Pex5p is a target for a ATP-utilizing component
mediating its release from the peroxisomal compartment. We do not know
the identity of this ATPase but it is obvious that Pex1p and/or Pex6p
(the only known peroxins containing ATP-binding domains (31-33)) are
good candidates. These two peroxins have been implicated in the
recycling of Pex5p back to the cytosol (13).
The results presented here allow us to propose a hypothetical model for
the Pex5p-mediated import of peroxisomal matrix proteins. Free Pex5p
(defined as stage 0 Pex5p) would bind newly synthesized PTS1-containing
proteins in the cytosol. This Pex5p-cargo protein complex (stage 1 Pex5p) would then diffuse to the peroxisomal membrane. We do not know
whether stage 1 Pex5p enters directly to the Pex14p-containing
docking-translocation complex at this stage or if it interacts first
with some other peroxisomal protein at the surface of the organelle
(e.g. Pex13p (34)): in this work only protease-protected
Pex5p species were characterized. Insertion of stage 1 Pex5p into the
Pex14p-containing complex results in stage 2 Pex5p. The
conformation/environment of stage 2 Pex5p is then changed, generating
stage 3 Pex5p. We do not know at what stage cargo proteins are released
from Pex5p. In principle, the conformation of the receptor domain of
Pex5p has to be modified to ensure that not only PTS1-containing
proteins are released but also prevented from re-binding. The stage
2/stage 3 transition could represent such a conformational
modification. The reason why stage 3 Pex5p is completely proteinase
K-resistant remains speculative. It is possible that the N terminus of
Pex5p moves toward the matrix side of the membrane as the PTS1 receptor
adopts the conformation required for its export from the peroxisomal compartment. Alternatively, the N terminus of stage 3 Pex5p is not
accessible to proteinase K as the result of a shielding effect performed by some other protein (e.g. the ATPase itself).
The fact that in purified rat liver peroxisomes only stage 2 Pex5p is
detected (see Fig. 8B (22)) together with the observation that in vitro import of Pex5p using purified organelles is
not inhibited by ATP It is obvious that this hypothetical model raises more questions than
it can answer. However, we believe that the results and the
experimental strategy presented in this work provide a good starting
point for an extensive mechanistic analysis of the Pex5p-mediated
protein import pathway. Similar studies using additional anti-peroxin
antibodies and mutant cell lines deficient in peroxisomal biogenesis
will surely provide valuable data.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C.
These fractions remain import-competent for at least 1 month.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S or no exogenous nucleotides. After 30 min at 26 °C, the
samples were put on ice and treated with proteinase K to degrade
nonimported Pex5p. The organelles were then isolated by centrifugation
and analyzed by SDS-PAGE. As shown in Fig.
1A, in the presence of
exogenous ATP, a small fraction of the 35S-labeled Pex5p
used in these experiments (1-5%) resisted the protease treatment
(lane 2). Most importantly, the vast majority of this
protease-resistant Pex5p population (hereafter referred to as stage 2 Pex5p; see "Discussion") does not represent the full-length
peroxin; a 2-kDa fragment was clipped from Pex5p by the action of
proteinase K. As shown before, this is exactly the proteolytic profile
of Pex5p present in highly pure rat liver peroxisomes (22), suggesting
that the in vitro synthesized Pex5p entered the peroxisomal
compartment. In the absence of exogenous nucleotides, two
protease-resistant Pex5p populations were observed (lane 1):
full-length Pex5p (hereafter referred to as stage 3 Pex5p) and a 2-kDa
truncated form (stage 2 Pex5p; see later). All attempts to degrade
stage 3 Pex5p, leaving stage 2 Pex5p intact, have failed (data not
shown); stage 3 Pex5p can only be degraded under conditions that
disrupt biological membranes (see below). The fact that
35S-labeled Pex5p acquires a protease-resistant location in
the absence of exogenous nucleotides could imply that the observed import does not require ATP. Alternatively, the low levels of ATP
present under these conditions (derived from both the PNS fraction and
the reticulocyte lysate; see "Experimental Procedures") may be
sufficient to promote this process. The result obtained when ATP
S is
used in an import assay favors this last possibility, the amount of
protease-resistant Pex5p is highly decreased (lane 3). A
similar inhibition on the import of 35S-labeled Pex5p was
observed in the presence of AMP-PNP or when the reaction components
were pretreated with apyrase (data not shown).
View larger version (25K):
[in a new window]
Fig. 1.
35S-Labeled Pex5p is in
vitro imported into peroxisomes. A, PNS
fractions were incubated with 35S-labeled Pex5p in import
buffer containing 1 mM ATP (lane 2), no
exogenous nucleotides (lane 1), or 1 mM ATP S
(lane 3). At the end of the incubation, samples were treated
with proteinase K to remove nonimported Pex5p and subjected to
SDS-PAGE. Lane I, 5% of the 35S-labeled Pex5p
used in each import reaction. The numbers at the
left indicate the molecular masses of the applied standards
in kDa. B, PNS fractions (1.5 mg of protein) subjected to an
import reaction with 35S-labeled Pex5p either in the
presence (panels A-D) or absence (panel
E) of 1 mM ATP were subjected to Nycodenz gradient
centrifugation. Samples shown in panels D and E
were treated with proteinase K before loading onto the gradients. After
centrifugation, the gradients were fractionated from the bottom
(lane 1) to the top (lane 12) of the tubes and
the distributions of 35S-labeled Pex5p (panels
C-E), rat liver Pex14p (panel B), and
several marker enzymes (panel A) were analyzed.
,
cytochrome c oxidase (mitochondria);
, esterase
(endoplasmic reticulum);
,
-glucocerebrosidase (lysosomes);
,
catalase (peroxisomes). Lane I, reticulocyte lysate
containing 35S-labeled Pex5p.
View larger version (34K):
[in a new window]
Fig. 2.
Functional/structural involvement of Pex14p
on the in vitro import of Pex5p. A,
PNS fractions (150 µg of protein) were preincubated with preimmune
IgGs (lane 1), anti-PMP70 IgGs (lane 2), or
anti-Pex14p IgGs (lane 3), and subjected to an import
reaction with 35S-labeled Pex5p in the absence of exogenous
nucleotides. After proteinase K treatment, the organelles were isolated
by centrifugation and subjected to SDS-PAGE. Lane I,
35S-labeled Pex5p (5% of the input in each lane).
B, two PNS fractions were incubated in import buffer either
in the presence (sample A) or absence (sample B)
of 35S-labeled Pex5p. After protease treatment,
35S-labeled Pex5p (2% of the amount used in sample A) was
added to sample B. After solubilization, aliquots corresponding to 600 µg of the initial PNS protein were subjected to immunoprecipitation
(IPP) using either anti-Pex14p IgGs (Pex14p, lanes
A and B) or preimmune IgGs (Preimmune;
lanes A and B). Lanes Total, aliquots
of samples A and B (corresponding to 100 µg of PNS protein) after the
solubilization step. IPP lanes were subjected to
autoradiography 6 times longer than lanes Total.
View larger version (36K):
[in a new window]
Fig. 3.
Stage 2 and stage 3 Pex5p in vitro
import kinetics. 35S-Labeled Pex5p was imported
into peroxisomes (600 µg of PNS protein) in the absence of exogenous
nucleotides. At the indicated times, aliquots (150 µg of PNS protein)
were removed and subjected to protease treatment. Lane I,
35S-labeled Pex5p (1% of the input).
View larger version (28K):
[in a new window]
Fig. 4.
GST-Pex5p blocks in vitro
import of Pex5p. A, GST-Pex5p (lanes
2, 4, and 6) or GST (lanes 1,
3, and 5) were added to import reactions in the
presence (+ATP) or absence of exogenous ATP
( ATP) at the indicated times. The total import time was 30 min. The import reactions were treated with proteinase K and the
organelles were analyzed by SDS-PAGE. B,
35S-labeled Pex5p was imported into peroxisomes either in
the presence (+ATP) or absence (
ATP) of
exogenous ATP for 18 min. GST-Pex5p or GST were then added to the
import reactions as indicated. Aliquots of the import reactions were
taken at the indicated time points, treated with proteinase K, and
analyzed by SDS-PAGE. Lane I, 35S-labeled Pex5p
(1% of the input).
S (1 mM final concentration) was added to the import
reaction (import of Pex5p in the presence of equimolar amounts of ATP
and ATP
S results in the formation of stage 2 and stage 3 Pex5p; data not shown). Aliquots of the import reaction were removed at several time points and analyzed. As shown in Fig.
5, stage 2 Pex5p can be transformed into
stage 3 Pex5p upon addition of ATP
S. In the second experiment,
in vitro synthesized Pex5p was subjected to an import
reaction in the absence of exogenous ATP. After 20 min, import of Pex5p
was stopped by adding GST-Pex5p and a chase in the presence of ATP (1 mM final concentration) was done. Stage 3 Pex5p disappears
rapidly from the peroxisomal compartment; the amount of stage 2 Pex5p
also decreases but in a slower way. Taken together, these experiments
suggest that stage 2 Pex5p is the precursor of stage 3 Pex5p and that
this population of peroxisomal Pex5p is capable of exiting the
organelle in an ATP-dependent process.
View larger version (35K):
[in a new window]
Fig. 5.
Relationship between stage 2 and stage 3 Pex5p. In vitro synthesized Pex5p was imported into
peroxisomes either in the absence (Pulse ATP) or presence
(Pulse +ATP) of exogenous ATP for 20 min. GST-Pex5p was then
added to both import reactions and, 7 min later, an aliquot was removed
(lanes 27'). ATP (Chase +ATP) or ATP
S
(Chase +ATP
S) were then added to the import
reactions, as indicated. Aliquots of the import reactions were taken 8 and 23 min later (lanes 35' and 50',
respectively). All the samples were treated with proteinase K and
analyzed by SDS-PAGE. Lane I, 35S-labeled Pex5p
(1% of the input).
S (lane 4) or in the absence of exogenous nucleotides
at 26 °C (lane 2). In sharp contrast, when a PNS fraction
is incubated at 26 °C in the presence of 1 mM ATP the
stage 2/stage 3 Pex5p ratio is largely increased (lane 3), suggesting that a significant amount of stage 3 Pex5p was exported from the peroxisomal compartment.
View larger version (51K):
[in a new window]
Fig. 6.
Export of endogenous Pex5p from the
peroxisomal compartment requires ATP hydrolysis. PNS fractions
(300 µg of protein) were incubated for 30 min at 0 (lane
1) or 26 °C (lanes 2-4) in import buffer B (see
"Experimental Procedures") containing no exogenous nucleotides
(lanes 1 and 2), 1 mM ATP (lane
3), or 1 mM ATP S (lane 4). Samples were
then divided into two equal aliquots. One-half was treated with
proteinase K, the organelles were isolated and subjected to Western
blotting analysis using anti-Pex5p (panel A) or
anti-catalase antibodies (panel B). The other half was
centrifuged and the organelle pellets (panel C) and the
supernatants (panel D) were analyzed by Western blotting
using an anti-Pex5p antibody. Note that in the ATP-treated sample, the
decrease in organelle-associated Pex5p (panel C, lane
3) is associated with a slight increase in the amount of soluble
Pex5p (panel D, lane 3).
View larger version (74K):
[in a new window]
Fig. 7.
The bulky portion of peroxisomal Pex5p is
exposed into the lumen of the organelle. 35S-Labeled
Pex5p was incubated with a PNS fraction (600 µg of protein) in import
buffer in the absence of exogenous ATP for 30 min. The organelles were
isolated by centrifugation and carefully resuspended in SEM buffer (see
"Experimental Procedures"). The organelles were divided into four
equal aliquots and proteinase K was added to three of these samples, as
indicated. One of these samples received Triton X-100
(TX-100) (lane 3); another was sonicated
immediately after addition of the protease (lane 4). After
trichloroacetic acid precipitation, all the samples were analyzed by
SDS-PAGE. Lane I, 35S-labeled Pex5p (1% of the
input). The numbers at the right indicate the
molecular masses of the applied standards in kDa.
View larger version (36K):
[in a new window]
Fig. 8.
The N terminus of stage 2 Pex5p is exposed
into the cytosol. A, GST-Pex5p (2 µg/lane) and
35S-labeled Pex5p (1 µl of reticulocyte lysate/lane) in
40 µl of Genenase I buffer (see "Experimental Procedures") were
incubated in the presence (lanes 2) or absence (lanes
1) of Genenase I. After trichloroacetic acid precipitation the
samples were analyzed by SDS-PAGE. The digestion of GST-Pex5p was
monitored by both Coomassie Blue staining (Coomassie) and
Western blotting using an anti-GST antibody (Anti-GST).
N-Pex5p, Pex5p cleaved at its N terminus;
GST', GST containing at its C terminus a few Pex5p amino
acid residues. The numbers at the left indicate
the molecular masses of the applied standards in kDa. B,
35S-labeled Pex5p was incubated with a PNS fraction in
import buffer in the presence of ATP. After import, the organelles were
isolated by centrifugation and carefully resuspended in SEM buffer.
One-half of this sample was treated with proteinase K (lanes
3 and 4); the other half received buffer only
(lanes 1 and 2). Phenylmethylsulfonyl fluoride
was then added to both samples, the organelles were reisolated by
centrifugation and resuspended in Genenase I buffer. Genenase I was
added as indicated. Protease treatment of purified rat liver
peroxisomes (Purified peroxisomes) was done in the same way.
After trichloroacetic acid precipitation, proteins were resolved by
SDS-PAGE and blotted onto a nitrocellulose membrane.
35S-Labeled Pex5p was detected by autoradiography;
peroxisomal rat liver Pex5p was detected using the anti-Pex5p
antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S) induced the release of a major fraction of endogenous
stage 3 Pex5p from peroxisomes. Thus, export of Pex5p from the
peroxisomal compartment requires ATP hydrolysis. Obviously, this
observation raises some questions concerning the need for ATP in Pex5p
import: if all the Pex5p-docking sites present in our PNS fractions
were occupied by the endogenous peroxin then import of
35S-labeled Pex5p would only be possible after peroxisomal
export of at least a fraction of the endogenous Pex5p. In the absence of experimental data addressing this possibility, our results do not
allow us to infer whether or not import of Pex5p requires ATP. However,
even if this proves to be the case, our data indicate that export of
Pex5p from the peroxisome is much more demanding on the ATP
concentration than its import into the organelle.
S and results in detection of stage 2 Pex5p
only,2 could support this interpretation. Finally, stage 3 Pex5p would be pulled from the peroxisomal membrane by an ATP-utilizing
component regenerating stage 0 Pex5p.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Joachim Rassow for critically reading the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by "Fundação para a Ciência e Tecnologia," Portugal, 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.), PRAXIS XXI/BD/16035/98 (to C. R.), and POCTI/BME/34648/99.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. Tel.:
351-226074900; Fax: 351-226099157; E-mail: jazevedo@ibmc.up.pt.
Published, JBC Papers in Press, October 30, 2002, DOI 10.1074/jbc.M209498200
2 A. M. Gouveia, C. P. Guimarães, M. E. Oliveira, C. Reguenga, C. Sá-Miranda, and J. E. Azevedo, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PTS, peroxisomal
targeting sequence;
MOPS, 3-(N-morpholino)propanesulfonic
acid;
GST, glutathione S-transferase;
PNS, post-nuclear
supernatant;
ATPS, adenosine 5'-O-(thiotriphosphate);
AMP-PNP, adenosine 5'-(
,
-imino)triphosphate.
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