INTRODUCTION

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To form a functional peroxisome, peroxisome membranes must be generated and the subsequent import of both membrane and matrix proteins must occur. Various studies have established that both peroxisomal membrane proteins (PMPs) and peroxisomal matrix proteins are made in the cytosol and delivered posttranslationally to the peroxisome (26). However, the similarities between PMP import and peroxisomal matrix protein import appear to end there. Peroxisomal matrix proteins are targeted to the peroxisome lumen via the presence of either a PTS1 or a PTS2 targeting signal (40). The PTS1 signal, which consists of a -SKL sequence (or a conserved variant thereof) at the extreme carboxy terminus of the protein (16), is present on the vast majority of peroxisomal matrix proteins. The PTS2 signal consists of the sequence R/K-L/V/I-X₅-H/Q-L/A near the amino terminus (41) and has so far been identified in only a handful of proteins. Both types of signals are recognized by specific receptors: Pex5p for the PTS1 signal (6, 29) and Pex7p for the PTS2 signal (27, 31). After binding to the receptor, matrix proteins are taken to the peroxisome surface and inserted into the organelle lumen through an as yet unknown mechanism. For Pex5p, it has been proposed that the receptor is then recycled back to the cytosol and can undergo further rounds of import (7). In contrast, PMPs lack PTS1 and PTS2 motifs (40), are imported independently of Pex5p and Pex7p (2), and require a distinct PMP-binding protein, Pex19p, for their import (19, 28, 34).

In addition to Pex5p, Pex7p, and Pex19p, a variety of other proteins required for peroxisome biogenesis have been identified (19). These include peroxins necessary for the biogenesis of peroxisome membranes (Pex3p), docking factors for the PTS receptors (Pex13p and Pex14p), proteins that act downstream of receptor docking but are required for translocation of proteins across the peroxisome membrane (Pex8p, Pex10p, and Pex12p), and other proteins whose functions are less clear (Pex1p, Pex2p, Pex4p, Pex6p, Pex17p, and Pex22p).

Despite the identification of so many components of peroxisome biogenesis, there is still little direct evidence as to the order in which these peroxins act. In the course of investigating the effects that different peroxins have on Pex5p, the PTS1 receptor, we observed that the loss of Pex1p or Pex6p in either yeast or human cells leads to an accelerated turnover of Pex5p and a significant drop in steady-state levels of Pex5p (7, 49). Koller et al. (25) have recently reported a similar phenotype for Pichia pastoris pex4 and pex22 mutants. Here we show that the phenotype of reduced Pex5p abundance in P. pastoris is unique to selected pex mutants and can be used to determine the epistatic relationships among the different peroxins. The results of our epistasis analysis suggest that Pex4p, Pex22p, Pex1p, and Pex6p act in the terminal steps of peroxisomal matrix protein import. This hypothesis is consistent with the reported phenotypes of P. pastoris pex1, pex6, and pex22 mutants, all of which contain peroxisomes and import detectable levels of peroxisomal matrix proteins. Similarly, we show here that the pex4 mutant also contains peroxisomes that are capable of peroxisomal matrix protein import.

	MATERIALS AND METHODS

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Yeast media. The various media used were based on those described earlier for P. pastoris (17). Specifically, YPD (1% yeast extract, 2% peptone, 2% dextrose) was used as rich medium. YPM (1% yeast extract, 2% peptone, 0.5% methanol) was used as rich methanol medium. YPOLT (1% yeast extract, 2% peptone, 0.18% oleic acid, 0.02% Tween 40) was used as rich oleic acid medium. SD + histidine (0.17% yeast nitrogen base without amino acids or ammonium sulfate, 0.5% ammonium sulfate, 0.1% L-histidine, 2% dextrose) was used as minimal-dextrose medium. SM + histidine + arginine (0.17% yeast nitrogen base without amino acids or ammonium sulfate, 0.5% ammonium sulfate, 0.1% L-histidine, 0.1% L-arginine, 0.5% methanol) was used as minimal-methanol medium. Sporulation medium (1% potassium chloride, 0.5% sodium acetate, 1% dextrose) was used to induce mating and sporulation. All cultures and plates were grown at 30°C.

Yeast strains. The yeast strains used in this study are presented in Table 1. The pex his4 strains were created by mating each pex mutant with SGY55 (4), sporulating the resultant diploids, and screening the haploid progeny for the pex his phenotype. The double-mutant strains created for this study were made as follows. The single-mutant strains in question were grown to saturation in YPD, and 0.5 ml of each was mixed and allowed to grow overnight on a YPD plate. On the following day, cells were scraped off the YPD plate, resuspended in 1 ml of sterile water, and plated on solid sporulation medium to induce allow mating. After 24 h, the sporulation plates were replica plated to minimal-methanol plates to select for diploids. Three days later, cells were transferred to fresh minimal-methanol plates and grown for another 3 days. The resulting diploid cells were then transferred to a 3-ml liquid YPD culture and grown overnight. The yeast cells from this culture were harvested, resuspended in 0.5 ml of sterile water, and seeded at high density onto solid sporulation medium. The cells were incubated for 5 days to allow the formation of a significant number of asci. The asci were then scraped from the plates, pelleted, and resuspended in 2 ml of sterile water. To separate asci, 650 µl of the resuspended pellet was incubated with 350 µl of 100% ethanol at room temperature for exactly 30 min. Cells were then plated at 10-fold serial dilutions to obtain single haploid spores. Spores were colony purified and assayed for growth on minimal-methanol medium. Spores that could not grow on methanol were then assayed by complementation analysis to distinguish the double-mutant progeny from the single-mutant progeny.

Plasmid construction and yeast transformations. The plasmids expressing the wild-type pex4 gene and the pex4-C133A mutant have been described previously (5). All bacterial manipulations were carried out with the Escherichia coli DH10B host (18). Purified plasmid DNA was introduced into the yeast strains by electroporation (5).

Electron and immunoelectron microscopy. For transmission electron microscopy, strains were grown in YPD to an optical density at 600 nm (OD₆₀₀) of 1.0, diluted to an OD₆₀₀ of 0.5, and incubated in YPM for 18 h to induce peroxisomal enzymes. Cells were fixed and processed as described previously (17). Samples were sectioned at 70 to 80 kV with a 35° angle diatome diamond knife and placed on Formvar-coated copper grids (Ted Pella, Inc.). The sections were poststained with 2% uranyl acetate and 0.3% lead citrate and observed on a Zeiss 10B transmission electron microscope.

Differential centrifugation, subcellular fractionation, and enzyme assays. All fractionation experiments were performed as previously described (5). For differential centrifugation, yeast cells were grown in YPD to mid-logarithmic phase, diluted to an OD₆₀₀ of 0.5, and shifted to rich oleic acid medium or minimal-methanol medium to induce proliferation of peroxisomal enzymes. After an 18- to 20-h incubation period, a postnuclear supernatant (PNS) of each strain was prepared. Organelles were then isolated by centrifugation of the PNS at 25,000 × g. Equal proportions of the organelle pellet and cytosolic supernatant were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed for the presence of the protein of interest by standard Western blot techniques. For subcellular fractionation, the 25,000 × g organelle pellet containing the mitochondria and peroxisomes (generated as described above) was fractionated over a 32 to 60% linear sucrose gradient as described previously (5). Fractions of 1 ml were collected from the bottom of the gradient and analyzed for catalase activity and succinate dehydrogenase activity.

RNA extraction and Northern blot analysis. The pex4 and pex10 strains were grown to an OD₆₀₀ of 0.6 in YPD, pelleted, and shifted to minimal-methanol medium. The methanol-induced cultures were incubated overnight at 30°C with agitation. On the following day, approximately 250 OD₆₀₀ units of cells were collected by centrifugation and total RNA was extracted as previously described (13). Poly(A)⁺ RNA was extracted from the total RNA by magnetic bead separation (Dynabeads; Dynal Inc.) in accordance with the manufacturer's directions.

Antibodies, protein extracts, and Western blotting. We have previously described the polyclonal antibodies recognizing P. pastoris Pex4p (5), Pex5p (15), and Pex10p (23). Anti-thiolase antibodies were raised against recombinant Saccharomyces cerevisiae thiolase and affinity purified. Secondary horseradish peroxidase-conjugated goat anti-rabbit antibodies were obtained from Sigma and Jackson ImmunoResearch.

Detection of initial synthesis of Pex5p. To detect Pex5p synthesis, the pex4 and pex10 strains were grown to log phase in YPD and subsequently shifted to minimal-methanol medium for 14 to 18 h. Cells were harvested and resuspended in 0.5 ml of minimal-methanol medium per 2.5 OD₆₀₀ units of cells. The resuspension was incubated at 30°C with agitation for 10 min, and the cells were then labeled by the addition of 75 µCi of [³⁵S]methionine per 0.5 ml (NEN EasyTag [³⁵S]methionine). After 2 to 5 min (2 min for the experiment shown), 20 µl of chase solution (0.5 M methionine, 0.5 M cysteine) was added per 0.5 ml. A 0.5-ml aliquot was then immediately transferred to 0.5 ml of ice-cold azide stop solution (0.04 M methionine, 0.04 M cysteine, 0.13% sodium azide). Cells were then pelleted and resuspended in 1 ml of ice-cold water. The cells were lysed, and total cellular protein was precipitated as outlined above. Protein pellets were resuspended in 30 µl of SDS-PAGE loading buffer (NaOH was added as needed to neutralize the sample) and boiled for 5 min to solubilize Pex5p. One milliliter of ice-cold dilution buffer (1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 50 mM Tris [pH 7.5]) plus protease inhibitors (Boehringer Mannheim Complete Tablets) was added, and the samples were allowed to sit on ice for 40 min to assist solubility. At this point, any nonsoluble debris were removed and a saturating amount of polyclonal anti-Pex5p antibody was added (diluted into 0.5 ml of dilution buffer plus protease inhibitors per sample, distributed to each sample from a single mixture). After incubation overnight at 4°C, a saturating amount of protein A agarose beads (Santa Cruz Biotechnology) was added and samples were incubated for an additional 90 min at 4°C with end-over-end rotation. The beads were then pelleted and washed five times in ice-cold wash buffer (0.1% Triton X-100, 0.02% SDS, 150 mM NaCl, 5 mM EDTA, 50 mM Tris [pH 7.5]). The beads were resuspended in 15 µl of SDS-PAGE buffer and boiled for 5 min, and the entirety of the sample was separated by SDS-PAGE. The gel was then soaked in 0.5 M sodium salicylate for 30 min, dried, and exposed to film.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Reduced abundance of the PTS1 receptor in cells lacking Pex4p activity. We have previously demonstrated that P. pastoris Pex4p is highly similar to known ubiquitin-conjugating enzymes, forms an adduct with ubiquitin in vivo, and requires its active-site cysteine (C133) for activity (5). Its likely function is therefore to ubiquitinate one or more target polypeptides. Given that the usual consequence of ubiquitination of target proteins is their degradation, we screened for peroxins that were stabilized by loss of Pex4p. We have yet to identify such a protein, but we did observe that pex4 cells contain extremely low levels of Pex5p, the PTS1 receptor (Fig. 1A). This phenotype has been previously observed by our lab for human and yeast cells lacking pex1 (7, 33) or pex6 (49) and has also been reported by Koller et al. for both the P. pastoris pex4 and pex22 mutants (25). Reduced Pex5p abundance is not a common phenotype of pex mutants (7), and the P. pastoris pex2-2, pex3-1, pex8-3, pex10, pex12, and pex13 mutants all contain normal levels of Pex5p (Fig. 1B). Normal Pex5p abundance was also observed in the pex14 and pex17 strains (data not shown). Although the pex1 and pex6-1 strains have reduced Pex5p levels, the phenotype is not as pronounced as in the pex4 or pex22 mutant. The average levels of Pex5p, compared to that of the wild type, were 35% for pex1, 48% for pex6-1, 10% for pex4, and 7% for pex22. However, it was not unusual for the levels of Pex5p to vary by 20% of the wild-type level from one trial to the next.

View larger version (46K): [in this window] [in a new window]   FIG. 1.   Loss of Pex4p results in reduced steady-state levels of Pex5p. (A and B) Equal amounts of protein were extracted from methanol-induced cells, separated by SDS-PAGE, and blotted with anti-Pex5p antibodies. (A) Levels of Pex5p in wild-type (WT) and pex4 cells. (B) Normal Pex5p levels in pex3, pex13, pex10, pex8, pex12, and pex2 strains. (C) Total cellular proteins extracted from pex4 strains carrying a wild-type PEX4 expression plasmid and a plasmid that expresses an active-site mutant of PEX4, C133A. Levels of Pex5p (left), Pex4p (middle), and the peroxisomal matrix enzyme thiolase (right) present in these samples were determined by immunoblotting with specific antibodies.

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Pex5p is synthesized normally in pex4 cells. To determine if the reduced level of Pex5p in the pex4 strain was due to reduced levels of PEX5 transcript, we performed Northern blot analysis on pex4 and pex10 cells. Poly(A)⁺ RNAs from pex4 and pex10 cells were separated by denaturing agarose gel electrophoresis, transferred to membranes, and sequentially hybridized to radiolabeled probes for the P. pastoris PEX5 and ACT1 (actin) genes (Fig. 2A). Quantitation of the PEX5 and ACT1 mRNA levels revealed that the PEX5:ACT1 ratio in the pex4 strain was 1.3 times that in the pex10 strain. Similar results were obtained in two additional independent trials (data not shown). Thus, the reduction of Pex5p levels in the pex4 strain cannot be due to decreased levels of PEX5 mRNA synthesis.

View larger version (46K): [in this window] [in a new window]   FIG. 2.   PEX5 mRNA and Pex5p are synthesized normally in pex4 cells. (A) Northern blot analysis of poly(A)+ RNAs from pex4 and pex10 cells. The two RNA samples were separated by denaturing gel electrophoresis, transferred to membranes, and sequentially hybridized to radiolabeled probes specific for the PEX5 (top) and ACT1 (bottom) genes. Quantification revealed that the PEX5:ACT1 hybridization signal was 1.3-fold higher in the pex4 strain than in the pex10 strain. Similar results were observed in two additional independent trials. (B) Synthesis of equivalent levels of Pex5p during pulse-labeling of pex4 and pex10 cells. Each strain was incubated in the presence of [35S]methionine for 2 min and lysed in alkali, and the Pex5p present in each sample was immunoprecipitated using excess anti-Pex5p antibodies. The immunoprecipitates were separated by SDS-PAGE, and the amount of Pex5p was determined by fluorographic exposure to X-ray film. Similar results were observed in nine additional trials of this experiment.

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Epistasis analysis places Pex4p late in peroxisomal matrix protein import. The variance in Pex5p stability displayed by different pex mutants allowed us to examine epistatic relationships among various pex mutants. An array of studies with both yeast (19, 21, 46, 48) and human (38) cells have established that PEX3 is required for synthesis of peroxisome membranes and that pex3 mutants are devoid of peroxisome-like structures. To test whether the reduced Pex5p levels seen in pex4 cells require the presence of peroxisome membranes and PEX3 function, we examined the levels of Pex5p in a pex3-1 pex4 double mutant. Total cellular protein was extracted from methanol-induced wild-type, pex5, pex3-1, pex4, and pex3-1 pex4 cells. The same amount of protein from each strain was then separated by SDS-PAGE and blotted with anti-Pex5p antibodies (Fig. 3A). In contrast to the low level of Pex5p detected in pex4 cells, the level of Pex5p observed in the pex3-1 pex4 double mutant was similar to those seen in the pex3-1 single-mutant and wild-type cells. These results demonstrate that Pex3p acts upstream of Pex4p. To visualize equivalency in loading, the protein remaining on the gel after transfer was stained with Coomassie blue (Fig. 3, 4, and 5).

View larger version (61K): [in this window] [in a new window]   FIG. 3.   The destabilization of Pex5p in the pex4 strain requires Pex3p, Pex13p, Pex14p, and Pex17p. For each panel, the strains were grown side by side and total cellular protein was extracted with alkali, separated by SDS-PAGE, and blotted with antibodies specific for Pex5p (top of each panel). Coomassie blue staining of each protein sample is also shown (bottom of each panel), and the quantity of Pex5p in each sample (as assessed by scanning densitometry of the resulting films) is shown below each lane. (A) Levels of Pex5p in wild-type (WT) cells; the pex5, pex3-1, and pex4 mutants; and the pex3-1 pex4 double mutant. (B) Levels of Pex5p in wild-type cells; the pex5, pex13, and pex4 mutants; and the pex4 pex13 double mutant. The lower blot in panel B shows levels of Pex5p in wild-type cells; the pex5, pex4, and pex14 mutants; the pex4 pex14 double mutant; the pex4 and pex17 mutants; and the pex4 pex17 double mutant.

View larger version (65K): [in this window] [in a new window]   FIG. 4.   Destabilization of Pex5p in the pex4 strain requires Pex10p, Pex12p, Pex8p, and Pex2p. For each panel, the strains were grown side by side and total cellular protein was extracted with alkali, separated by SDS-PAGE, and blotted with antibodies specific for Pex5p (top of each panel). Coomassie blue staining of each protein sample is also shown (bottom of each panel), and the quantity of Pex5p in each sample (as assessed by scanning densitometry of the resulting films) is shown below each lane. (A) Levels of Pex5p in wild-type (WT) cells; the pex5, pex4, and pex10 mutants; and the pex4 pex10 double mutant. The lower blot in panel A shows levels of Pex5p in wild-type cells; the pex5, pex12, and pex4 mutants, and the pex4 pex12 double mutant. (B) Levels of Pex5p in wild-type cells; the pex5, pex8-3, and pex4 mutants; the pex4 pex8-3 double mutant; the pex2-2 and pex4 mutants; and the pex2-2 pex4 double mutant.

View larger version (55K): [in this window] [in a new window]   FIG. 5.   Pex1p and Pex6p act downstream of Pex10p and upstream of Pex4p and Pex22p. For each panel, the strains were grown side by side and total cellular protein was extracted with alkali, separated by SDS-PAGE, and blotted with antibodies specific for Pex5p (top of each panel). Coomassie blue staining of each protein sample is also shown (bottom of each panel), and the quantity of Pex5p in each sample (as assessed by scanning densitometry of the resulting films) is shown below each lane. (A) Levels of Pex5p in wild-type (WT) cells; the pex5, pex1, and pex10 mutants; the pex1 pex10 double mutant; the pex6-1 and pex10 mutants; and the pex6-1 pex10 double mutant. The lower blot in panel A shows levels of Pex5p in wild-type cells; the pex5, pex4, and pex22 mutants; the pex4 pex22 double mutant; the pex10 and pex22 mutants; and the pex10 pex22 double mutant. (B) Levels of Pex5p in wild-type cells; the pex5, pex1, and pex4 mutants; the pex1 pex4 double mutant; the pex6-1 and pex4 mutants; and the pex6-1 pex4 double mutant. (C) Levels of Pex5p in wild-type cells; the pex5, pex1, pex6-1, and pex22 mutants; and the pex1 pex22 and pex6-1 pex22 double mutants.

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Pex1p, Pex6p, and Pex22p also act late in peroxisomal matrix protein import. The phenotype of Pex5p instability is also shared by the pex22 mutant and to lesser extents by the pex1 and pex6-1 mutants. While a previous study has established that Pex22p interacts physically with Pex4p (25), there is no evidence linking PEX4 or PEX22 function with PEX1 or PEX6 function. Therefore, we tested whether these mutants might also act late in peroxisomal matrix protein import. To do this, we generated double-mutant combinations of pex1, pex6-1, and pex22 with pex10, which blocks matrix protein import downstream of PTS receptor docking (3). Pex5p levels were examined in pex1 pex10, pex6-1 pex10, and pex10 pex22 double mutants, and in each case the levels of Pex5p were similar to that of the pex10 single mutant (Fig. 5A). The pex4 pex22 double mutant was also examined and found to have low Pex5p levels that were similar to those of both single mutants (Fig. 5A). The slight increase seen in the quantitation (6% for the double mutant versus undetectable for the single mutants) is not outside the sensitivity limit of the quantitation method.

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Peroxisomes of pex4 mutants contain residual levels of peroxisomal matrix proteins. Several lines of evidence indicate that peroxisomal matrix protein import involves the cycling of PTS receptors between the cytoplasm and peroxisome, suggesting that the terminal step in matrix protein import is the release of PTS receptors back to the cytoplasm (42). Cells defective in this step would be expected to contain recognizable peroxisomes, import PMPs normally, and import residual levels of peroxisomal matrix proteins.

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View larger version (135K): [in this window] [in a new window]   FIG. 6.   Transmission electron microscopy reveals the presence of small, matrix-containing peroxisomes in pex4 cells. Wild-type (WT) (A and B) and pex4 (C and D) cells were induced in methanol medium and processed for transmission electron microscopy. M, mitochondria; N, nucleus; P, peroxisome; V, vacuole. Bar, 1.0 µm.

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View larger version (113K): [in this window] [in a new window]   FIG. 7.   Immunocryoelectron microscopy demonstrates that pex4 cells contain small, electron-dense peroxisomes. (A) Peroxisomes (p) of wild-type cells labeled with antibodies specific for Pex10p, shown here by immunogold labeling of the peroxisome membrane. Note the position of gold particles at the peroxisome membrane (shown by arrowheads) and the electron-dense nature of the protein-rich peroxisome matrix. (B to F) Peroxisomes of pex4 cells are also labeled with antibodies specific for Pex10p and contain an electron-dense, granular matrix. However, their radius is only 10% of the radius of wild-type peroxisomes, indicating that their volume may be only 1/1,000 of that of the wild-type peroxisome. Bar, 0.1 µm.

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View larger version (26K): [in this window] [in a new window]   FIG. 8.   The pex4 mutant contains peroxisomes of normal density. Wild-type (WT), pex4, and pex10 cells were induced in methanol medium, a PNS was generated from each strain, and peroxisomes and mitochondria were collected by differential centrifugation. The resulting three organelle pellets were then separated by sucrose density gradient centrifugation. The same amount of each fraction was assayed for catalase (a PTS1-containing peroxisomal enzyme) activity (black bars), succinate dehydrogenase (mitochondrial enzyme) activity (grey bars), and density (data not shown). Enzyme activities are plotted as percentages of the total activity in the gradient. The densities of all of the gradient profile were similar, with the peak peroxisomal fraction of wild-type cells migrating at a density of 1.19 to 1.21 g/cm3 and the peak peroxisomal fraction of pex4 cells migrating at a density of 1.18 to 1.20 g/cm3.

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View larger version (31K): [in this window] [in a new window]   FIG. 9.   The pex4 mutant imports significant amounts of the PTS2 marker enzyme thiolase but less than wild-type (WT) or pex5 cells. Wild-type, pex4, pex5, and pex10 cells were induced to proliferate peroxisomal proteins, and a PNS was generated from each strain. This was then separated into an organelle pellet (p) and a cytosolic supernatant (s) by centrifugation at 25,000 × g. The same proportion of each fraction was separated by SDS-PAGE and blotted with antibodies specific for the PTS2-targeted enzyme thiolase (top) and the integral PMP Pex10p (bottom).

Subcellular distribution of Pex5p in the pex4 strain. A previous study has established that P. pastoris Pex5p is a predominantly cytosolic, partly peroxisomal protein in wild-type cells (15). The hypothesis that Pex4p plays an important role in the recycling of Pex5p back to the cytoplasm predicts that loss of Pex4p should result in accumulation of Pex5p at the peroxisome. We examined the distribution of Pex5p in wild-type, pex4, pex5, and pex10 cells. Strains were induced in methanol medium, and postnuclear supernatants (PNS) were prepared and separated into an organelle pellet and a cytosolic supernatant. Equal proportions of these fractions were assayed Pex5p by Western blot analysis (Fig. 10). Virtually all of the Pex5p present in pex4 cells was associated with the organelle pellet, a stark contrast to the predominantly cytosolic distribution in wild-type and pex10 cells.

View larger version (21K): [in this window] [in a new window]   FIG. 10.   Most Pex5p is present in the organelle fraction of pex4 cells. Wild-type (WT), pex4, pex5, and pex10 cells were induced in methanol medium, and a PNS was generated from each strain. This was then separated into an organelle pellet (p) and a cytosolic supernatant (s) by centrifugation at 25,000 × g. The same proportion of each fraction was separated by SDS-PAGE and blotted with antibodies specific for Pex5p.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Genetic studies of peroxisome biogenesis have led to the identification of more than 20 different peroxins, each required for normal peroxisome biogenesis. Studies of these factors and of peroxisomal protein import mechanisms have shown that PMP import and peroxisomal matrix protein import occur through separate pathways, with each process controlled by a distinct set of genes. As all peroxisomal matrix proteins are translated in the cytosol, their proper localization is dependent on the action of the PTS receptors. Studies on Pex5p, the PTS1 receptor, indicate that it is a predominantly cytoplasmic, partly peroxisomal protein (6, 8, 15, 45) and may cycle between the cytoplasm and the peroxisome (7). Such results have inspired models of peroxisomal matrix protein import in which peroxins are required not only for the translocation of proteins through the peroxisome membrane but also to facilitate the movement of PTS receptors through their cycles. Such a view of peroxisomal matrix protein import suggests the existence of PTS receptor docking factors, matrix protein translocation factors, and PTS receptor recycling factors.

Within the context of this model, determining the order of action for different peroxisome biogenesis factors should be extremely useful for determining their functions. Obviously, biochemical approaches can be used to determine the order of peroxin action. Such studies have led to current models in which Pex14p acts in PTS receptor docking in peroxisomal matrix protein import (42) and Pex8p (32), Pex10p, and Pex12p (3) act downstream of receptor docking in peroxisomal matrix protein import. However, epistasis analysis is ideally suited for determining the order of gene action, provided that there are defining subphenotypes that can be used as markers of different steps in the process being studied. In this study, we used the subphenotype of reduced Pex5p abundance to examine the epistatic relationships among 12 different pex genes.

We have previously established that reduced Pex5p abundance is a reproducible phenotype for human pex1- and pex6-deficient cell lines but is not observed in human pex2-, pex7-, pex10-, pex12-, and pex16-deficient cells (7, 49). Furthermore, these studies revealed that the reduced abundance of Pex5p in pex1- and pex6-deficient cells was due to an increased rate of Pex5p degradation. Koller et al. recently reported that Pex5p abundance is reduced in P. pastoris pex4 and pex22 mutants (25). Here we show that Pex5p abundance is normal in P. pastoris pex2-2, pex3-1, pex8-3, pex10, pex12, pex13, pex14, and pex17 mutants and that the reduction in Pex5p abundance is more severe in pex4 and pex22 mutants than in pex1 and pex6 mutants. Furthermore, we demonstrated that pex4 cells have normal PEX5 mRNA abundance and are able to synthesize Pex5p. That pex4 and pex10 mutants have similar rates of Pex5p synthesis but different steady-state levels of Pex5p represents strong evidence that Pex5p is degraded at an accelerated rate in pex4 cells. These results are consistent with the increased rate of Pex5p degradation in human pex1- and pex6-deficient cells (7, 49).

Although technical difficulties in chasing labeled methionine from P. pastoris cells prevented us from measuring Pex5p stability in the pex4 and pex10 mutant strains, we were still able to use the variance in Pex5p abundance to order the pex mutants relative to one another by epistasis analysis. Our data show that Pex4p acts after the peroxisome membrane synthesis factor Pex3p, after the PTS receptor docking factors Pex13p and Pex14p, after the putative protein translocation factors Pex8p, Pex10p, and Pex12p, and after the other peroxins Pex2p and Pex17p. Furthermore, we found that Pex1p, Pex6p, and Pex22p also act downstream of Pex10p and that Pex1p and Pex6p act upstream of both Pex4p and Pex22p. This result suggests a model in which Pex1p, Pex4p, Pex6p, and Pex22p all act downstream of matrix protein translocation (Fig. 11).

View larger version (38K): [in this window] [in a new window]   FIG. 11.   Model of peroxisomal matrix protein import. PTS-containing proteins are bound by their receptor after translation in the cytoplasm (step 1). The mechanism by which the receptor-bound matrix protein is transported to the peroxisome membrane remains unclear (step 2). The receptor-bound matrix protein then docks on the surface of the peroxisome membrane via interactions with the docking factors Pex13p, Pex14p, and possibly Pex17p (step 3). Following docking, the matrix protein must dissociate from the receptor and be translocated across the peroxisome membrane into the matrix space (step 4). It is not clear whether the receptor follows the cargo into the lumen or remains on the cytosolic surface of the peroxisome. However, in either scenario, the receptor is subsequently released from the translocation pore and recycled back to the cytoplasm to undergo further rounds of import (step 5).

The placement of Pex4p very late in peroxisomal matrix protein import is consistent with the cellular phenotypes of pex4 mutants. Electron microscopy studies revealed that pex4 cells still have peroxisomes and that these peroxisomes resemble those of wild-type cells in all respects but their overall size. Peroxisomes of pex4 cells had the clustered, cuboidal, electron-dense appearance of normal methanol-induced P. pastoris peroxisomes, but their diameter was 10 times less. Biochemical studies revealed that peroxisomes of pex4 cells import wild-type levels of the PMP Pex10p and import reduced but significant levels of both PTS1- and PTS2-targeted peroxisomal matrix proteins. In contrast, pex10 cells displayed more severe peroxisomal matrix protein import defects, indicating that the peroxisomal matrix protein import defects of the pex4 mutant are unusually mild. The phenotypes of the pex4 mutant are not what we would expect for loss of a peroxin that plays an essential role in peroxisome membrane synthesis, PMP import, PTS receptor docking, or peroxisomal matrix protein translocation. However, they are consistent with the loss of a peroxin that participates in PTS receptor recycling, which is thought to be the final step in peroxisomal matrix protein import. This hypothesis for Pex4p function predicts that Pex5p should be trapped at or in the peroxisome in pex4 cells, and it is interesting that 75% of the Pex5p in pex4 cells is present in the organelle fraction (compared to only 17% in wild-type cells).

An analysis of Hansenula polymorpha Pex4p also concluded that Pex4p participates in the recycling of Pex5p to the cytoplasm (44). That study also found that Pex5p accumulates on or in peroxisomes in the absence of Pex4p. However, they also found that the peroxisomal protein import defect of pex4 cells could be partially suppressed by overexpressing PEX5 (44). Interestingly, H. polymorpha pex4 mutants import PTS2 proteins normally (44) whereas S. cerevisiae and P. pastoris pex4 mutants display a reproducible defect in PTS2 protein import (47). The mild PTS2 protein import defect and the severe reduction in Pex5p abundance in P. pastoris pex4 cells raise the possibility that the phenotypes of pex4 cells is simply due to reduced Pex5p abundance. However, the PTS2 protein import defect of pex4 cells is more severe than that of pex5 cells. Also, we tested whether PEX5 overexpression could rescue the growth defects of pex4 cells but failed to see any evidence of phenotypic rescue. Thus, it is unlikely that reduced Pex5p abundance can explain all of the phenotypes of pex4 cells.

Koller et al. (25) have recently reported that Pex22p physically interacts with Pex4p, that pex22 mutants show severely reduced Pex5p levels, and that Pex22p is required for Pex4p abundance. We previously established that Pex4p is peripherally associated with the outer surface of the peroxisome membrane (5), and Koller et al. (25) speculated that Pex22p may function as a docking site for Pex4p on the peroxisome membrane. Thus, it is was not surprising that our epistasis analysis placed Pex22p after Pex1p, Pex6p, and Pex10p and, by deduction, after all of the other peroxins we examined, except Pex4p. These multiple lines of evidence connect Pex22p and Pex4p at a terminal step in peroxisomal matrix protein import.

Our epistasis studies indicate that Pex1p and Pex6p also act late in peroxisomal matrix protein import, though upstream of Pex4p and Pex22p. The action of Pex1p and Pex6p at a common point in peroxisome biogenesis is altogether expected, given that genetic interactions have been described for PEX1 and PEX6 and that physical interactions have been reported for Pex1p and Pex6p (10, 12, 24). Furthermore, results that place Pex1p and Pex6p at a late step in peroxisomal matrix protein import are consistent with the phenotypes of pex1 and pex6 mutants in most species. In humans (33, 36, 49) and the yeasts P. pastoris (20, 39), H. polymorpha (24), and S. cerevisiae (19), cells lacking pex1 or pex6 contain numerous peroxisomes that are competent for PMP import. Studies of the yeasts P. pastoris (20, 39) and H. polymorpha (24) and human cells (33, 49) have also shown that peroxisomes of pex1- and pex6-deficient cells import residual levels of peroxisomal matrix proteins. Thus, the pex1 and pex6 mutants, like the pex4 and pex22 mutants, display phenotypes that we might expect from the loss of receptor recycling factors. The epistasis analysis presented here clearly places P. pastoris Pex1p and Pex6p downstream of Pex10p, a protein that acts downstream of receptor docking in peroxisomal matrix protein import, supporting the hypothesis that Pex1p and Pex6p act late in peroxisomal matrix protein import. However, studies of the yeast Yarrowia lipolytica have led Rachubinski and colleagues to propose a different model in which Pex1p and Pex6p participate in peroxisome membrane biogenesis (43). We have no model that can explain these differences in results or interpretation, and it may be that the roles of Pex1p and Pex6p are quite different in Y. lipolytica.

How the Pex1p-Pex6p step of peroxisome biogenesis relates to the subsequent step defined by Pex4p-Pex22p remains to be determined. However, the fact that reduced Pex5p abundance is observed in cells lacking any of these four factors indicates that there may be a functional connection between these two steps. Pex1p and Pex6p are a pair of interacting AAA ATPases, and it is well established that these proteins participate in the formation, organization, and/or dissociation of protein complexes (30). It is perhaps worthwhile to consider the possibilities that Pex1p and Pex6p can prepare or present substrates for ubiquitination by a Pex4p-Pex22p complex and that the ubiquitination event has a positive effect on Pex5p stability. However, the possibilities for how Pex4 and Pex22p protect Pex5p from degradation are numerous. For example, it could involve the destruction of a protein that degrades Pex5p or a modification of Pex5p that protects it from degradation by some general proteolysis machinery. Elucidation of the molecular mechanisms by which Pex1p, Pex6p, Pex4p, and Pex22p impact the stability of Pex5p is clearly a challenge but must be pursued if we are to understand the roles of these peroxins and the process of PTS receptor recycling.