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© The Rockefeller University Press,
0021-9525/2000//931 $5.00
The Journal of Cell Biology, Volume 148, Number 5,
, 2000 931-944
Original Article |
Pex19 Binds Multiple Peroxisomal Membrane Proteins, Is Predominantly Cytoplasmic, and Is Required for Peroxisome Membrane Synthesis
Peroxisomes are components of virtually all eukaryotic cells. While much is known about peroxisomal matrix protein import, our understanding of how peroxisomal membrane proteins (PMPs) are targeted and inserted into the peroxisome membrane is extremely limited. Here, we show that PEX19 binds a broad spectrum of PMPs, displays saturable PMP binding, and interacts with regions of PMPs required for their targeting to peroxisomes. Furthermore, mislocalization of PEX19 to the nucleus leads to nuclear accumulation of newly synthesized PMPs. At steady state, PEX19 is bimodally distributed between the cytoplasm and peroxisome, with most of the protein in the cytoplasm. We propose that PEX19 may bind newly synthesized PMPs and facilitate their insertion into the peroxisome membrane. This hypothesis is supported by the observation that the loss of PEX19 results in degradation of PMPs and/or mislocalization of PMPs to the mitochondrion.
Key Words: Zellweger syndrome • organelle • peroxisome biogenesis receptor • protein import
© 2000 The Rockefeller University Press
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Introduction |
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The formation of peroxisomes must involve the generation of the peroxisome membrane, the targeting and insertion of peroxisomal membrane proteins (PMPs) into this membrane, and the transport of peroxisomal matrix proteins to and across this same bilayer. Of these processes, most is known about peroxisomal matrix protein import. Proteins destined for the peroxisome matrix are encoded by nuclear genes, synthesized in the cytoplasm (Lazarow and Fujiki 1985), and contain either of two peroxisomal targeting signals, PTS1 and PTS2 (Subramani 1993). Import of these proteins is dependent upon PEX5 and PEX7, the receptors for the PTS1 and PTS2 (McCollum et al. 1993; Marzioch et al. 1994), and occurs in an ATP-dependent manner (Imanaka et al. 1987). The fact that the PTS receptors are predominantly cytoplasmic proteins (Marzioch et al. 1994; Dodt et al. 1995) has suggested models of import in which the receptors act as shuttles, delivering newly synthesized matrix proteins to the peroxisome (Braverman et al. 1995; Dodt and Gould 1996; Hettema et al. 1999). These models also invoke a variety of other peroxins that catalyze the translocation of proteins across the peroxisome membrane and return the PTS receptors to the cytoplasm.
Although peroxisomes play critical roles in numerous metabolic pathways they are not essential for cell viability. Therefore, it is not surprising that defects in peroxisome biogenesis can cause human diseases by interfering with peroxisomal metabolic processes. Zellweger syndrome (ZS) and related diseases are characterized by defects in peroxisome biogenesis and the loss of multiple peroxisomal metabolic processes (Lazarow and Moser 1995). Consistent with the complexity of organelle biogenesis, mutations in any of at least 12 different PEX genes can cause ZS. In most cases, loss of PEX gene activity disrupts peroxisomal matrix protein import, but has no effect on the synthesis of peroxisome membranes or import of peroxisomal membrane proteins (PMPs; Santos et al. 1988; Chang et al. 1999b). However, there are a few rare ZS patients who lack detectable peroxisomal structures, indicating that the genes defective in these patients, PEX3, PEX16, and PEX19, are involved in synthesis of the peroxisome membrane or import of PMPs (Matsuzono et al. 1999; South and Gould 1999; South, S.T., K.A. Sacksteder, S.J. Gould, manuscript in preparation). Studies in yeast have identified up to 20 PEX genes that are required for peroxisome biogenesis, and again, most mutants appear competent for PMP import (Hettema et al. 1999).
PMPs are synthesized on free polyribosomes and posttranslationally inserted into the peroxisome membrane (Fujiki et al. 1984; Imanaka et al. 1996). However, the similarities between PMP import and peroxisomal matrix protein import appear to end there. PMPs lack recognizable PTS1- or PTS2-like features (Subramani 1993), most pex mutants that disrupt matrix protein import have no effect on PMP import (Chang et al. 1999b), and PMP import appears to be ATP-independent (Diestelkotter and Just 1993). To better understand the molecular mechanisms of PMP import and the synthesis of the peroxisome membrane, we examined the biochemical properties of PEX19, a protein that is required for the synthesis of peroxisome membranes in both human and yeast cells (Gotte et al. 1998; Matsuzono et al. 1999). We report here that human PEX19 is a predominantly cytoplasmic PMP-binding protein, and we discuss the relevance of our findings for PMP import and peroxisome biogenesis.
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Materials and Methods |
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We have previously described the plasmids pcDNA3-PEX10, pcDNA3-PEX10myc (Warren et al. 1998), pcDNA3-PEX11β, pcDNA3-PEX11βmyc (Schrader et al. 1998), pcDNA3-PEX12, pcDNA3-PEX12myc (Chang et al. 1997), pcDNA3-PEX13 (Bjorkman et al. 1998), and pcDNA3-PECI (Geisbrecht et al. 1999). The plasmids pcDNA3-PEX3, pcDNA3-PEX14, pcDNA3-PMP34, pcDNA1/Amp-PMP70, pcDNA3-ALDP, and pcDNA3-ALDR all contained the full-length ORF for the gene downstream of the cytomegalovirus promoter and T7 promoter in the appropriate vector. Myc tags were appended to these genes and to human PMP22, PMP24, PEX11
, and PEX13 by amplification using primers designed to append Asp718 and BamHI sites at the 5' and 3' ends, respectively, and these fragments were cloned upstream of and in-frame with a 12–amino acid myc tag in the plasmid pcDNA3myc (Yahraus et al. 1996). The plasmids pJL59W-PEX10, pJL59W-PEX11β, pJL59W-PEX12, and pJL59W-PEX13 contain the ORF of each gene in-frame with the GAL4 DNA binding domain of pJL59W. Control plasmids used in the blot overlay experiments (SP6-Tim23, SP6-Aac2, SP6-MIR1, T7-IBV-M, and T7-VSV-G) were obtained from Drs. Robert Jensen and Carolyn Machamer (The Johns Hopkins University School of Medicine, Baltimore, MD). The plasmids pcDNA3-PMP70myc, pcDNA3-
C395PMP70myc, pcDNA3-C476PMP70myc, pcDNA3-C535PMP70myc, pcDNA3-C558PMP70myc, pcDNA3-C578PMP70myc, and pcDNA3-C598PMP70myc were created by amplifying the appropriate regions of the human PMP70 cDNA with oligonucleotides designed to append an Asp718 site immediately upstream of the start codon (GGTACCATG; start codon underlined) and a BamHI site after the last codon of the desired ORF. These fragments were cleaved with Asp718 and BamHI and inserted between the Asp718 and BamHI sites of pcDNA3myc. The plasmids pcDNA3-N119PEX11βmyc, pcDNA3-N180PEX11βmyc, pcDNA3-N210PEX11βmyc, pcDNA3-C231PEX14myc, and pcDNA3-C270PEX14myc were all created in a similar fashion. Bacterial vectors designed to express 6xHis-PEX14, mutant forms of 6xHis-PEX14, 6xHis-PTE1, and 6xHis-XECI were created by transferring the coding regions of each clone into the bacterial expression vector, pT7-His (Geisbrecht et al. 1999). For all plasmids, any regions generated by PCR were sequenced to ensure the absence of any unintended mutations.
Dihybrid Analysis and Blot Overlay Assays
The yeast strain BY3168 (Vidal et al. 1996) was used for all dihybrid assays. Pairs of plasmids, one encoding the appropriate binding domain (BD) fusion, the other encoding the appropriate AD fusion, were used to transform the yeast to leucine and tryptophan prototrophy. After selection, the modified strains were incubated at 30°C for 2 d, transferred to BA-85 membranes (Schleicher and Schuell), grown for an additional 2 d, and assayed for β-galactosidase activity.
For blot overlay assays, all in vitro transcription/translation reactions were performed using TNT rabbit reticulocyte lysates (Promega). 5 µg of 6xHis-PEX19 and a nonspecific control protein (XECI, a nuclear enoyl-CoA isomerase) were resolved by SDS-PAGE electrophoresis and transferred to PVDF membrane. The membrane was blocked for 2 h at room temperature in buffer A (500 mM Tris-HCl, pH 7.5, 50 mM NaCl, 100 mM sodium acetate, 100 mM KCl, 1 mM DTT, 5 mM MgCl2, 1 mM EDTA, 0.3% Tween 20, 0.1 mM ZnCl2, 5% milk, 0.1 M methionine). The membrane was incubated for 2 h at room temperature with 5 ml of buffer A containing 35S-labeled PMPs that had been synthesized in vitro, washed, and exposed to film. For far Western analysis, membranes containing 5 µg of each protein were blocked as before, incubated with 10 ml of buffer A containing 2 µg of purified 6xHis-PEX14, washed, and probed with affinity-purified anti–PEX14 antibodies.
For quantitative analysis of PEX19-PEX14 binding, 35S-labeled 6xHis-PEX14 was synthesized in vitro, mixed with bacterially expressed 6xHis-PEX14, and the resulting mixture was applied at various concentrations to membranes containing 100 ng of immobilized PEX19. After the binding reaction, the filters were washed briefly and the amount of PEX14 bound to PEX19 was determined using a PhosphorImager (MacBAS; Fuji). For competition assays, a small amount of labeled PEX14 or PEX3 (
1–10 nM) was mixed with the indicated concentrations of purified recombinant PEX14, PTE1, or mutant forms of PEX14 and incubated with immobilized PEX19 using the blot overlay technique. The labeled PMP bound was detected using a PhosphorImager. All 6xHis-tagged proteins were expressed and purified from bacteria as described (Geisbrecht et al. 1999).
Transfections, Immunofluorescence, and Antibody Production
Cell lines were cultured and transfected by electroporation and processed for indirect immunofluorescence 4 h after transfection as described (Chang et al. 1997, Chang et al. 1999b). Affinity-purified PEX19 antibodies were used for all immunofluorescence experiments. Monoclonal mouse anti-myc antibodies were obtained from the tissue culture supernatant of the hybridoma 1-9E10 (Evan et al. 1985), guinea pig anti–PMP70 antibodies were raised against the COOH-terminal 18 amino acids of human PMP70, and secondary antibodies were obtained from commercial sources. Antibodies directed against PEX19 were generated by immunization of rabbits with an MBP-PEX19 fusion protein and were affinity-purified against immobilized 6xHis-PEX19. The human skin fibroblast cell line GM5756-T was used for the expression of proteins in normal human fibroblasts. The cell line PBD399, which corresponds to the same Zellweger syndrome patient cell line examined by Matsuzono et al. 1999, was used for the functional complementation studies and for assessing PMP fates in the absence of PEX19. The functional complementation studies were performed by transfecting PBD399 cells by electroporation as described (Chang et al. 1997) with the plasmids pcDNA3, pcDNA3-PEX19, and pcDNA3-PEX19/C296A. 3 d after transfection, the cells were processed for indirect immunofluorescence using established protocols (Dodt and Gould 1996) and antibodies specific for catalase, which is a peroxisomal matrix protein. Relative rescue activity was calculated as described (Chang and Gould 1998).
Rat Liver Fractionation, Differential Centrifugation, Differential Permeabilization, and Immunoblotting
Preparation of rat liver postnuclear supernatant, separation of the peripheral nervous system by Nycodenz centrifugation and assay of marker enzymes were performed as described previously using a 15–45% Nycodenz gradient (Mihalik 1992). For differential centrifugation analysis, a rat liver was homogenized, a postnuclear supernatant was generated, and then spun at 25,000 g for 30 min. This supernatant was removed and subjected to further centrifugation at 200,000 g for 1 h. Equal proportions of the resulting homogenate, supernatants, and pellets were processed for immunoblot with anti–PEX19 antibodies. For the differential release assay, GM5756-T fibroblasts were harvested by trypsinization, washed, and divided into seven equal fractions. The cells were gently resuspended in 100 µl of STE (10 mM Tris, 1 mM EDTA, 250 mM sucrose) that was adjusted to various concentrations of digitonin (0, 50, 100, 200, 300, and 400 µg/ml) or 400 µg/ml digitonin and 1% Triton X-100. After a 10-min incubation on ice, clarified supernatants were generated by centrifugation and analyzed by immunoblot. All immunoblots were performed using HRP-conjugated secondary antibodies and chemiluminescent detection (Amersham).
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, PEX14, and PEX16), negative results in this assay can be caused by a variety of nonspecific problems, such as poor expression, improper folding, and failure to target to the nucleus, none of which were controlled for in this study. Furthermore, it should be noted that the yeast two-hybrid assay is generally not useful for assessing interactions with integral membrane proteins (Drees 1999) such as the PMPs tested here.
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We also examined the specificity of PEX19 for integral PMPs (Fig. 1 B). We found that PEX19 does not bind peroxisomal matrix proteins, shown here by its inability to bind peroxisomal enoyl-CoA isomerase (PECI; Geisbrecht et al. 1999), nor does it bind to itself. PEX19 was also unable to bind to integral membrane proteins of other organelles, including an integral plasma membrane protein (VSV-G; Adams and Rose 1985), an integral Golgi membrane protein (IBV-M; Boursnell et al. 1984), and three integral mitochondrial membrane proteins (Tim23; Dekker et al. 1993), Aac2 (Lawson and Douglas 1988), and Mir1 (Murakami et al. 1990). It should be noted that these control membrane proteins were added at approximately the same concentration as the PMPs that did bind to PEX19.
To test whether PEX19-PMP binding was direct, we expressed and purified a small amount of soluble PEX14 from bacteria (it was the only PMP that we were able to purify in soluble form). We performed the blot overlay assay using recombinant PEX14 rather than in vitro translated PEX14. The ability of PEX19 to bind recombinant PEX14 was determined by blotting with affinity-purified anti–PEX14 antibodies. This far Western assay revealed that recombinant PEX19 was indeed able to bind purified PEX14 in the absence of any other factors (Fig. 1 B).
The blot overlay assay was also used to examine the thermodynamic parameters of PEX19-PMP binding. Immobilized PEX19 was incubated with various concentrations of recombinant PEX14 spiked with 35S-labeled PEX14, and the relative amounts bound were quantitated using a PhosphorImager (Fig. 2 A). Binding was saturated at concentrations of 3 µM and above in three independent experiments. The actual dissociation constants calculated in the three trials were 350, 800, and 500 nM, suggesting a PEX14-PEX19 dissociation constant of 500 nM. To assess the specificity of PEX19 binding, we tested the ability of peroxisomal thioesterase (PTE1; Jones et al. 1999), a peroxisomal matrix protein, and PEX14 to compete 35S-labeled PMPs from binding to PEX19 (Fig. 2 B). Excess unlabeled PEX14 effectively competed labeled PEX14 from PEX19, but excess PTE1 had no effect on PEX19-PEX14 binding. Specific competition was also observed when PTE1 and PEX14 were assayed for their ability to compete with labeled PEX3 for PEX19 binding.
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myc (Schrader et al. 1998), PEX13myc, PEX16myc (South and Gould 1999), PMP24myc (Reguenga et al. 1999), and PMP22myc (Diestelkotter and Just 1993; data not shown).
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Targeting Elements of PMPs Retain Interaction with PEX19
The ability of PEX19 to bind PMPs of diverse function implicates PEX19 in the biogenesis of PMPs. One obvious possibility is that PEX19 may interact with regions of PMPs that are involved in targeting to peroxisomes. To test whether this might be true, we examined the regions of several human PMPs that are required for targeting to peroxisomes and tested whether they retained the ability to bind to PEX19. The integral PMPs, PMP70, PEX11β, and PEX14, were chosen for these studies. COOH-terminal truncations of PMP70, each of which carried a COOH-terminal myc epitope tag, were expressed in human fibroblasts and their subcellular distributions were determined by immunofluorescence. The first 124 amino acids of PMP70 were sufficient to direct it into peroxisomes (Fig. 4B and Fig. C). This same segment of PMP70 is efficiently misdirected to the nucleus in cells that overexpress NLS/PEX19 (Fig. 4D and Fig. E). The first 101, 81, or 61 amino acids of PMP70 are also able to mediate peroxisomal targeting but only at reduced efficiency. These shorter pieces of PMP70 were localized to both peroxisomes and mitochondria, as shown here for C598 PMP70, the smallest of the three proteins (Fig. 4F and Fig. G). The positive identification of the tubulovesicular structures as mitochondria was confirmed by colocalization with a mitochondrial marker (MitoTracker; data not shown). These three pieces of PMP70 retained their ability to interact with PEX19, as seen by the misdirection of C598 PMP70 to the nucleus in cells overexpressing NLS-PEX19 (Fig. 4H and Fig. I).
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PEX19 is a farnesylated protein and it is possible that PEX19 might use its farnesyl group to associate with peroxisome membranes. If true, the homogenization and permeabilization experiments may have released PEX19 from peroxisomes without affecting the distribution of other peroxisomal proteins. Because it is virtually impossible to control directly for this possibility, we decided instead to address this issue indirectly by determining whether farnesylation was critical for PEX19 function. PEX19 is farnesylated via the cysteine residue at position 296, and we generated a PEX19 mutant in which this cysteine is replaced by an alanine (PEX19/C296A) and is, therefore, unable to accept the farnesyl moiety. We transfected a PEX19-deficient human fibroblast cell line with pcDNA3, pcDNA3-PEX19, and pcDNA3-PEX19/C296A, and assessed the ability of each plasmid to restore peroxisome biogenesis in these cells (Fig. 7, D–F). Transfection with the empty vector had no effect, expression of WT PEX19 led to rescue of peroxisome biogenesis in 50% of the cells, and expression of PEX19/C296A led to restoration of peroxisome biogenesis in a similarly high number of the cells. Quantitation of relative rescue activities revealed that the C296A mutation had only a mild effect on PEX19 activity, reducing it to 80% the activity of wild-type PEX19. Therefore, it appears that farnesylation has an ancillary effect on PEX19 function and that PEX19 functions well in the absence of the farnesyl moiety.
PMP Fates in the Absence of PEX19
It has been established previously that PMP70 is present at greatly reduced levels in PEX19-deficient mammalian cells, and that this is caused by an increased rate of PMP70 degradation (Kinoshita et al. 1998; Matsuzono et al. 1999). We examined the fates of additional PMPs in PEX19-deficient cells to assess the role of PEX19 in the biogenesis of other PMPs. Immunofluorescence studies showed that normal human fibroblasts contain hundreds of PMP70-containing peroxisomes but, as previously demonstrated, PEX19-deficient cells lack detectable PMP70-containing structures (Fig. 8A and Fig. B). Similar results were observed when these cells were stained with antibodies specific for the integral PMPs PEX13 and PEX11β (Fig. 8, C–F). However, not all PMPs were undetectable in PEX19-deficient cells. Immunoblot experiments revealed that the integral PMP PEX14 was present at similar levels in PEX19-deficient cells as in other PEX mutants (data not shown) and was easily detectable in these cells by immunofluorescence. However, the PEX14 in these cells was not present in peroxisome-like structures or other small vesicles, but was instead mislocalized to the mitochondria (Fig. 9, A–C). Similar mitochondrial mislocalization was observed for myc-tagged derivatives of several integral PMPs that were introduced by transfection. These included the integral PMPs PEX12 (Fig. 9, D–F) and ALDP (Fig. 9, G–I). Similar mitochondrial mislocalization was observed when PEX13 and PEX11β were overexpressed in the PEX19-deficient cells (data not shown). Snyder et al. 1999 recently reported that minute, PEX3-containing minivesicles may exist in PEX19-deficient cells of the yeast Pichia pastoris. We could not directly test whether similar structures exist in human PEX19-deficient cells because we lack antibodies of sufficient titer to PEX3. However, myc-tagged PEX3, like the other PMPs we tested, was easily detected in peroxisomes of normal cells, but was mislocalized to mitochondria of PEX19-deficient cells (Fig. 9, J–L).
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PEX19 Is a Broad Specificity PMP-binding Protein
Using a blot overlay assay, we found that PEX19 binds to a broad spectrum of PMPs with diverse functions in peroxisome biogenesis (PEX3, PEX12, PEX13, and PEX14) and metabolite transport (PMP70, PMP34, and ALDR). The binding activity of PEX19 appeared to be specific for PMPs as PEX19 failed to bind peroxisomal matrix proteins or integral membrane proteins that reside in the plasma, Golgi, or mitochondrial membranes. The fact that PEX19-PMP binding could be assayed in vitro allowed us to further characterize the nature of PEX19-PMP binding. Using PEX14 as a test PMP, we found that the PEX19–PMP interaction displayed a dissociation constant of 500 nM, which is well within the range expected for a reversible protein–protein interaction. For comparison, it is interesting to note that the complex between PTS1-containing peroxisomal matrix proteins and PEX5, the PTS1 receptor, also has a dissociation constant of 500 nM (Terlecky et al. 1995).
The interaction between PEX19 and these same seven PMPs was also tested in an independent PEX19-binding assay in which a nuclear localization signal was appended to PEX19 and the resulting NLS/PEX19 protein was assayed for its ability to mislocalize newly synthesized PMPs to the nucleus. In this second assay, PEX19 interacted with 14 human PMPs, including the same set of 7 PMPs that PEX19 bound to in the blot overlay assay (PEX3, PEX12, PEX13, PEX14, PMP70, PMP34, and ALDR) as well as 7 more PMPs (PEX10, PEX11, PEX11β, PEX16, ALDP, PMP22, and PMP24). Furthermore, we observed that PEX19 interacted with four human PMPs in the yeast two-hybrid assay: PEX10, PEX11β, PEX12, and PEX13. All told, we observed interaction of PEX19 with nine human PMPs in at least two independent assays (PEX12 and PEX13 in all three assays; PEX3, PEX14, PMP70, PMP34, ALDR in the blot overlay and nuclear mislocalization assays; and PEX11β and PEX10 in the two-hybrid and nuclear mislocalization assays) and 14 human PMPs altogether. It should be noted that ours are not the first evidence that PEX19 can bind PMPs, as previous reports have demonstrated that S. cerevisiae PEX19 binds PEX3 (Gotte et al. 1998) and that P. pastoris PEX19 binds PEX3 and PEX10 (Snyder et al. 1999). Although we did not detect interaction of PEX19 with several PMPs assayed in the yeast two-hybrid assay (PEX2, PEX3, PEX11, PEX14, and PEX16), these results are difficult to interpret in the absence of controls for the expression, folding, and nuclear targeting of the various GAL4BD-PMP fusion proteins.
PMP Targeting Elements Retain the Ability to Bind PEX19
The interaction of human PEX19 with a diverse array of human PMPs, the ability of NLS/PEX19 to draw newly synthesized, but not mature, PMPs into the nucleus, and the inability of PEX19-deficient cells to import PMPs all indicate that PEX19 plays an important role in PMP biogenesis, perhaps in the recognition of PMP targeting signals or as a chaperone for newly synthesized PMPs. If true, we would expect that regions of PMPs that were sufficient for targeting to peroxisomes would retain the ability to bind PEX19, and we tested this for three integral PMPs, PMP70, PEX11β, and PEX14.
We found that the first 61 amino acids of PMP70 (<10% of the protein) are sufficient for peroxisomal localization and that this same segment of PMP70 retained the ability to interact with PEX19. The analysis of PEX11β provided similar results, with the COOH-terminal 49 amino acids of PEX11β being sufficient for both targeting and interaction with PEX19. We also used a more quantitative method for the analysis of PEX14-PEX19 binding. Targeting studies revealed that the first 147 amino acids of PEX14 targeted to peroxisomes, though weakly, and bound to PEX19 with a much lower affinity than full-length PEX14. We also found that the first 108 amino acids of PEX14 could not bind to PEX19, and that this fragment of PEX14 could not localize to peroxisomes. Taken together, these results are consistent with the hypothesis that targeting elements of PMPs retain the ability to bind PEX19.
It is important to note that the targeting elements described in this paper cannot be equated with the minimal targeting signals for these PMPs; thus, the question of whether PEX19 binds to PMP targeting signals remains to be addressed. However, our inability to identify the PMP targeting signals in PMP70 and PEX11β was not due to a lack of effort. For both PMP70 and PEX11β, we were unable to identify regions smaller than 50–60 amino acids that could target to peroxisomes, and we have encountered similar problems during searches for targeting signals in the PMPs PMP34, PEX12, and PEX13 (data not shown). Such problems were not encountered during the search for targeting signals in peroxisomal matrix proteins (Subramani 1993), and this difference is worth discussing. It is our opinion that the difficulty in identifying concise PMP targeting signals lies in the nature of protein targeting assays. Conventional targeting assays obviously measure the targeting abilities of the test proteins, but it is generally not appreciated that they also measure the retention of the protein in the organelle after the targeting event. In the case of peroxisomal matrix proteins, the retention is provided by the passive barrier of the peroxisome membrane and the assay, therefore, reflects just the targeting information of the protein. In contrast, it is difficult to envision how PMPs could be retained unless they were inserted into the peroxisome membrane, a process that is unlikely to be passive. Thus, biologically defined PMP targeting signals are likely to include signals for two potentially distinct processes: targeting, which may involve the recognition by a PMP receptor, and retention, which may require insertion of the protein into the peroxisome membrane. The targeting elements we identified in PMP70, PEX11β, and PEX14 all contained a putative transmembrane domain, and it will be interesting to determine the nature of the PEX19-binding sites in these and other PMP targeting elements.
In addition to the test proteins we used for determining whether peroxisomal targeting elements retained the ability to bind to PEX19, it is necessary to discuss three proteins that we did not use in these particular experiments. The first is Candida boidinii PMP47. If we define a PMP targeting signal as the minimal regions that are sufficient for targeting to peroxisomes and for which the functional elements are known, the only PMP targeting signal in the literature that meets this criteria is the mPTS of C. boidinii PMP47 (Dyer et al. 1996). We considered testing whether PEX19 recognized this mPTS, but we found that this signal is not sufficient for peroxisomal membrane targeting in human cells (Gould, S.J., unpublished observations) and it was, therefore, unclear what benefit could come from such an analysis. The other two proteins that deserve mention are PEX3 and PEX16. We detected interactions between PEX19 and these two PMPs. Like any other integral PMPs we might have considered using PEX3 or PEX16 to test whether PEX19-binding sites reside within their PMP targeting elements. This is particularly true for PEX3 since previous studies have localized a low efficiency PMP targeting element to within the first 40 amino acids of this PMP (Kammerer et al. 1998; Soukupova et al. 1999). However, we consciously avoided using these PMPs for these particular experiments because they, like PEX19, are required for peroxisome membrane biogenesis along with PEX19 (Hettema et al. 1999; Matsuzono et al. 1999; South and Gould 1999; South, S.T., K.A. Sacksteder, and S.J. Gould, unpublished observations). Thus, they may interact with PEX19 in multiple places and for multiple purposes. For example, it is possible that PEX19 could interact with targeting elements during the biogenesis of PEX3 and PEX16, but also bind to other regions of PEX3 and/or PEX16 as it mediates the biogenesis of other PMPs. This is not to say that an analysis of targeting signals and PEX19-binding sites in PEX3 and PEX16 is unimportant. Rather, it reflects our view that such studies will be extremely important not only for what they will tell us about general PEX19–PMP interactions, but also for the information they will provide on the roles of these three proteins in the biogenesis of peroxisome membranes. In contrast, the analysis of targeting elements and PEX19-binding sites in PMPs that are not required for peroxisome membrane synthesis, such as PMP70 and PEX11β, are likely to yield data that relates only to the general role of PEX19 binding in PMP biogenesis.
PEX19 Is Bimodally Distributed between Cytoplasm and Peroxisomes
To better understand how PEX19 facilitates peroxisome membrane biogenesis, we assessed its distribution within the cell. Subcellular fractionation, differential centrifugation, and differential permeabilization experiments all indicated that there is a large population of cytosolic, soluble PEX19, in addition to a peroxisomal pool of PEX19. Our results are consistent with previously published data that mammalian PEX19 is both cytoplasmic (James et al. 1994; Matsuzono et al. 1999) and peroxisomal (James et al. 1994; Kammerer et al. 1997; Matsuzono et al. 1999). A similar bimodal distribution has been observed for yeast forms of PEX19 (Gotte et al. 1998; Snyder et al. 1999). Quantitation of the PEX19 levels in cytosolic and peroxisomal fractions of our rat liver gradient fractions suggest that >95% of PEX19 may be cytoplasmic.
One potential caveat to our PEX19 distribution data is related to PEX19 farnesylation. It is formally possible that PEX19 utilizes its farnesyl moiety for association with peroxisomes and that our homogenization and permeabilization procedures released PEX19 from peroxisomes without disturbing peroxisome integrity in general. To assess the general relevance of farnesylation to PEX19 function we used site-directed mutagenesis to eliminate the farnesylation site. WT PEX19 and the resulting PEX19/C296A mutant were assayed for their ability to restore peroxisome biogenesis in PEX19-deficient cells, and we found that the mutant was almost fully active (80% of WT activity). High activity also has been reported for an analogous CAAX box mutant of P. pastoris PEX19 (Snyder et al. 1999). These studies strongly suggest that farnesylation has an ancillary rather than a central role in PEX19 function, and makes it unlikely that PEX19 uses farnesylation as its primary means for associating with peroxisomes. Our results are based on the analysis of four independent, sequence-confirmed PEX19/C296A clones, and we have no explanation for the previous report that a C296S mutant of human PEX19 had no activity (Matsuzono et al. 1999).
Implications for PMP Import and Peroxisome Biogenesis
In this report, we found that PEX19 binds to a diverse array of PMPs, including metabolite transporters and peroxins, and that regions of PMPs which are sufficient for targeting to peroxisomes retain the ability to interact with PEX19. We also observed that PEX19 is a predominantly cytoplasmic, partly peroxisomal protein, and that loss of PEX19 results in the absence of detectable peroxisomal structures, the destabilization of many integral PMPs, and the mislocalization of other PMPs to the mitochondrion. One simple model that could explain these data is that PEX19 plays an important role in the biogenesis of PMPs. For instance, the bimodal distribution of PEX19 may represent a steady state view of a cycling PMP-binding protein that facilitates the early steps in PMP biogenesis, either as a chaperone or as a PMP receptor. Such a model provides many questions for future studies, such as whether PEX19 is indeed a PMP receptor, whether PEX19 binds PMPs in the cytoplasm before their import, whether PEX19 cycles between cytoplasm and peroxisome, and whether PEX19 has additional roles when present at the peroxisome membrane.
Existing data on peroxisome biogenesis in human cells are consistent with the ability of cells to generate peroxisomes in two ways, either by growth and division of preexisting peroxisomes or by synthesis from an as yet unidentified preperoxisomal vesicle. In this model (South and Gould 1999), peroxisomes are defined as vesicles that contain a normal or nearly normal complement of PMPs (regardless of matrix protein content), whereas preperoxisomal vesicles are defined as vesicles that either lack PMPs altogether or have only one or a small number of PMPs. In our examination of PEX19-deficient cells, we did not see PMPs present in any vesicles reminiscent of peroxisomes or in any smaller vesicles. Instead, they were either completely absent or mislocalized to the mitochondrion. Although we did not find any evidence for the early remnants reported in pex19 mutants of P. pastoris by Snyder et al. 1999, it is possible that we could have missed such structures with the techniques that we employed.
Recent studies from yeast have suggested that the ER may contribute to peroxisome synthesis (for reviews see Kunau and Erdmann 1998; Titorenko and Rachubinski 1998), and the ER could, in fact, represent preperoxisomal vesicles of our model. However, the hypothesis that the ER plays a direct and essential role in peroxisome biogenesis still lacks evidence from multiple experimental systems that would be necessary for its general acceptance. The results of this study do not directly address this question, but it is worth noting that our results offer no clear support for an ER role in peroxisome membrane synthesis. For instance, we failed to observe accumulation of any PMPs in the ER in the absence of PEX19, a result that would be predicted for PMPs that transit through the ER before their import into peroxisomes, as has been suggested for PEX3 and PEX16 (for review see Kunau and Erdmann 1998; Titorenko and Rachubinski 1998). Also, the fact that PEX19 is a predominantly cytoplasmic PMP-binding protein with an important role in PMP biogenesis does lend some support to the model that PMPs, or at least most PMPs, are synthesized on free polyribosomes and imported directly from the cytoplasm into peroxisomes (Lazarow and Fujiki 1985).
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Acknowledgments |
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This work was support by grants from the National Institutes of Health and the March of Dimes to S.J. Gould (DK45787, HD10981, and MOD FY93/94-0514).
Submitted: 27 May 1999
Revised: 5 January 2000
Accepted: 21 January 2000
Abbreviations used in this paper: AD, activation domain; BD, binding domain; NLS, nuclear localization signal; ORF, open reading frame; PMP, peroxisomal membrane protein; ZS, Zellweger syndrome.
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