Pex8p: an intraperoxisomal organizer of the peroxisomal import machinery

Peroxisomes transport folded and oligomeric proteins across their membrane. Two cytosolic import receptors, Pex5p and Pex7p, along with approximately 12 membrane-bound peroxins participate in this process. While interactions among individual peroxins have been described, their organization into functional units has remained elusive. We have purified and defined two core complexes of the peroxisomal import machinery: the docking complex comprising Pex14p and Pex17p, with the loosely associated Pex13p, and the RING finger complex containing Pex2p, Pex10p, and Pex12p. Association of both complexes into a larger import complex requires Pex8p, an intraperoxisomal protein. We conclude that Pex8p organizes the formation of the larger import complex from the trans side of the peroxisomal membrane and thus might enable functional communication between both sides of the membrane.     

Molecular anatomy of the peroxin Pex12p: ring finger domain is essential for Pex12p function and interacts with the peroxisome-targeting signal type 1-receptor Pex5p and a ring peroxin, Pex10p

The three peroxin genes, PEX12, PEX2, and PEX10, encode peroxisomal integral membrane proteins with RING finger at the C-terminal part and are responsible for human peroxisome biogenesis disorders. Mutation analysis in PEX12 of Chinese hamster ovary cell mutants revealed a homozygous nonsense mutation at residue Trp263Ter in ZP104 cells and a pair of heterozygous nonsense mutations, Trp170Ter and Trp114Ter, in ZP109. This result and domain mapping of Pex12p showed that RING finger is essential for peroxisome-restoring activity of Pex12p but not necessary for targeting to peroxisomes. The N-terminal region of Pex12p, including amino acid residues at positions 17-76, was required for localization to peroxisomes, while the sequence 17-76 was not sufficient for peroxisomal targeting. Peroxins interacting with RING finger of Pex2p, Pex10p, and Pex12p were investigated by yeast two-hybrid as well as in vitro binding assays. The RING finger of Pex12p bound to Pex10p and the PTS1-receptor Pex5p. Pex10p also interacted with Pex2p and Pex5p in vitro. Moreover, Pex12p was co-immunoprecipitated with Pex10p from CHO-K1 cells, where Pex5p was not associated with the Pex12p-Pex10p complex. This observation suggested that Pex5p does not bind to, or only transiently interacts with, Pex10p and Pex12p when Pex10p and Pex12p are in the oligomeric complex in peroxisome membranes. Hence, the RING finger peroxins are most likely to be involved in Pex5p-mediated matrix protein import into peroxisomes.     

Distinct Modes of Ubiquitination of Peroxisome-targeting Signal Type 1 (PTS1) Receptor Pex5p Regulate PTS1 Protein Import

Peroxisome targeting signal type-1 (PTS1) receptor, Pex5p, is a key player in peroxisomal matrix protein import. Pex5p recognizes PTS1 cargoes in the cytosol, targets peroxisomes, translocates across the membrane, unloads the cargoes, and shuttles back to the cytosol. Ubiquitination of Pex5p at a conserved cysteine is required for the exit from peroxisomes. However, any potential ubiquitin ligase (E3) remains unidentified in mammals. Here, we establish an in vitro ubiquitination assay system and demonstrate that RING finger Pex10p functions as an E3 with an E2, UbcH5C. The E3 activity of Pex10p is essential for its peroxisome-restoring activity, being enhanced by another RING peroxin, Pex12p. The Pex10p·Pex12p complex catalyzes monoubiquitination of Pex5p at one of multiple lysine residues in vitro, following the dissociation of Pex5p from Pex14p and the PTS1 cargo. Several lines of evidence with lysine-to-arginine mutants of Pex5p demonstrate that Pex10p RING E3-mediated ubiquitination of Pex5p is required for its efficient export from peroxisomes to the cytosol and peroxisomal matrix protein import. RING peroxins are required for both modes of Pex5p ubiquitination, thus playing a pivotal role in Pex5p shuttling.     

The mammalian peroxin Pex5pL, the longer isoform of the mobile peroxisome targeting signal (PTS) type 1 transporter, translocates the Pex7p.PTS2 protein complex into peroxisomes via its initial docking site, Pex14p

In mammals, two isoforms of the peroxisome targeting signal (PTS) type 1 receptor Pex5p, i.e. Pex5pS and Pex5pL with an internal 37-amino acid insertion, have previously been identified. Expression of either type of Pex5p complements the impaired PTS1 import in Chinese hamster ovary pex5 mutants, but only Pex5pL can rescue the PTS2 import defect noted in a subgroup of pex5 mutants such as ZP105. In this work, we found that Pex5pL directly interacts with the PTS2 receptor Pex7p, carrying its cargo PTS2 protein in the cytosol. Pex5pL, but not Pex5pS, mediated the binding of PTS2 protein to Pex14p by translocating Pex7p, demonstrating that Pex5pL plays a pivotal role in peroxisomal PTS2 import. Pex5p was localized mostly in the cytosol in wild-type CHO-K1 and Pex14p-deficient mutant cells, whereas it accumulated in the peroxisomal remnants in cell mutants defective in Pex13p or the RING family peroxins such as Pex2p and Pex12p. Furthermore, overexpression of Pex14p, but not Pex10p, Pex12p, or Pex13p, caused accumulation of Pex5p in peroxisomal membranes, with concomitant interference with PTS1 and PTS2 import. Therefore, Pex5p carrying the cargoes most likely docks with the initial site (Pex14p) in a putative import machinery, subsequently translocating to other components such as Pex13p, Pex2p, Pex10p, and Pex12p.     

Membrane association of the cycling peroxisome import receptor Pex5p

Peroxisomal proteins carrying a peroxisome targeting signal type 1 (PTS1) are recognized in the cytosol by the cycling import receptor Pex5p. The receptor-cargo complex docks at the peroxisomal membrane where it associates with multimeric protein complexes, referred to as the docking and RING finger complexes. Here we have identified regions within the Saccharomyces cerevisiae Pex5p sequence that interconnect the receptor-cargo complex with the docking complex. Site-directed mutagenesis of the conserved tryptophan residue within a reverse WXXXF motif abolished two-hybrid binding with the N-terminal half of Pex14p. In combination with an additional mutation introduced into the Pex13p-binding site, we generated a Pex5p mutant defective in a stable association not only with the docking complex but also with the RING finger peroxins at the membrane. Surprisingly, PTS1 proteins are still imported into peroxisomes in these mutant cells. Because these mutations had no significant effect on the membrane binding properties of Pex5p, we examined yeast and human Pex5p for intrinsic lipid binding activity. In vitro analyses demonstrated that both proteins have the potential to insert spontaneously into phospholipid membranes. Altogether, these data strongly suggest that a translocation-competent state of the PTS1 receptor enters the membrane via protein-lipid interactions before it tightly associates with other peroxins.     

Pex12p of Saccharomyces cerevisiae is a component of a multi-protein complex essential for peroxisomal matrix protein import

We have isolated the Saccharomyces cerevisiae pex12-1 mutant from a screen to identify mutants defective in peroxisome biogenesis. The pex12delta deletion strain fails to import peroxisomal matrix proteins through both the PTS1 and PTS2 pathway. The PEX12 gene was cloned by functional complementation of the pex12-1 mutant strain and encodes a polypeptide of 399 amino acids. ScPex12p is orthologous to Pex12 proteins from other species and like its orthologues, S. cerevisiae Pex12p contains a degenerate RING finger domain of the C3HC4 type in its essential carboxy-terminus. Localization studies demonstrate that Pex12p is an integral peroxisomal membrane protein, with its NH2-terminus facing the peroxisomal lumen and with its COOH-terminus facing the cytosol. Pex12p-deficient cells retain particular structures that contain peroxisomal membrane proteins consistent with the existence of peroxisomal membrane remnants ("ghosts") in pex12A null mutant cells. This finding indicates that pex12delta cells are not impaired in peroxisomal membrane biogenesis. In immunoisolation experiments Pex12p was co-purified with the RING finger protein Pex10p, the PTS1 receptor Pex5p and the docking proteins for the PTS1 and the PTS2 receptor at the peroxisomal membrane, Pex13p and Pex14p. Furthermore, two-hybrid experiments suggest that the two RING finger domains are sufficient for the Pex10p-Pex12p interaction. Our results suggest that Pex12p is a component of the peroxisomal translocation machinery for matrix proteins.     

Pex7p and Pex20p of Neurospora crassa function together in PTS2-dependent protein import into peroxisomes

Recruiting matrix proteins with a peroxisomal targeting signal type 2 (PTS2) to the peroxisomal membrane requires species-specific factors. In Saccharomyces cerevisiae, the PTS2 receptor Pex7p acts in concert with the redundant Pex18p/Pex21p, whereas in Yarrowia lipolytica, Pex20p might unite the function of both S. cerevisiae peroxins. Herein, the genome of the filamentous fungus Neurospora crassa was analyzed for peroxin-encoding genes. We identified a set of 18 peroxins that resembles that of Y. lipolytica rather than that of S. cerevisiae. Interestingly, proteins homologous to both S. cerevisiae Pex7p and Y. lipolytica Pex20p exist in N. crassa. We report on the isolation of these PTS2-specific peroxins and demonstrate that NcPex20p can substitute for S. cerevisiae Pex18p/Pex21p, but not for ScPex7p. Like Pex18p, NcPex20p did not bind PTS2 protein or the docking proteins in the absence of ScPex7p. Rather, NcPex20p was required before docking to form an import-competent complex of cargo-loaded PTS2 receptors. NcPex7p did not functionally replace yeast Pex7p, probably because the N. crassa PTS2 receptor failed to associate with Pex18p/Pex21p. However, once NcPex7p and NcPex20p had been coexpressed, it proved possible to replace yeast Pex7p. Pex20p and Pex18p/Pex21p are therefore true orthologues, both of which are in need of Pex7p for PTS2 protein import.     

A role for the peroxin Pex8p in Pex20p-dependent thiolase import into peroxisomes of the yeast Yarrowia lipolytica

Peroxins are proteins required for peroxisome assembly. The cytosolic peroxin Pex20p binds directly to the beta-oxidation enzyme thiolase and is necessary for its dimerization and peroxisomal targeting. The intraperoxisomal peroxin Pex8p has a role in the import of peroxisomal matrix proteins, including thiolase. We report the results of yeast two-hybrid analyses with various peroxins of the yeast Yarrowia lipolytica and characterize more fully the interaction between Pex8p and Pex20p. Coimmunoprecipitation showed that Pex8p and Pex20p form a complex, while in vitro binding studies demonstrated that the interaction between Pex8p and Pex20p is specific, direct, and autonomous. Pex8p fractionates with peroxisomes in cells of a PEX20 disruption strain, indicating that Pex20p is not necessary for the targeting of Pex8p to peroxisomes. In cells of a PEX8 disruption strain, thiolase is mostly cytosolic, while Pex20p and a small amount of thiolase associate with peroxisomes, suggesting the involvement of Pex8p in the import of thiolase after docking of the Pex20p-thiolase complex to the membrane. In the absence of Pex8p, peroxisomal thiolase and Pex20p are protected from the action of externally added protease. This finding, together with the fact that Pex8p is intraperoxisomal, suggests that Pex20p may accompany thiolase into peroxisomes during import.     

Pex18p is constitutively degraded during peroxisome biogenesis

Pex18p and Pex21p are structurally related yeast peroxins (proteins required for peroxisome biogenesis) that are partially redundant in function. One or the other is essential for the import into peroxisomes of proteins with type 2 peroxisomal targeting sequences (PTS2). These sequences bind to the soluble PTS2 receptor, Pex7p, which in turn binds to Pex18p (or Pex21p or possibly both). Here we show that Pex18p is constitutively degraded with a half-time of less than 10 min in wild-type Saccharomyces cerevisiae. This degradation probably occurs in proteasomes, because it requires the related ubiquitin-conjugating enzymes Ubc4p and Ubc5p and occurs normally in a mutant lacking the Pep4p vacuolar protease. The turnover of Pex18p stops, and Pex18p accumulates to a much higher than normal abundance in pex mutants in which the import of all peroxisomal matrix proteins is blocked. This includes mutants that lack peroxins involved in receptor docking at the membrane (Deltapex13 or Deltapex14), a mutant that lacks the peroxisomal member of the E2 family of ubiquitin-conjugating enzymes (Deltapex4), and others (Deltapex1). This stabilization in a variety of pex mutants indicates that Pex18p turnover is associated with its normal function. A Pex18p-Pex7p complex is detected by immunoprecipitation in wild type cells, and its abundance increases considerably in the Deltapex14 peroxisome biogenesis mutant. Cells that lack Pex7p fail to stabilize and accumulate Pex18p, indicating an important role for complex formation in the stabilization. Mono- and diubiquitinated forms of Pex18p are detected in wild-type cells, and there is no Pex18p turnover in a yeast doa4 mutant in which ubiquitin homeostasis is defective. These data represent, to the best of our knowledge, the first instance of an organelle biogenesis factor that is degraded constitutively and rapidly.     

Peroxisome remnants in pex3delta cells and the requirement of Pex3p for interactions between the peroxisomal docking and translocation subcomplexes

During peroxisomal matrix protein import, the peroxisomal targeting signal receptors recognize cargo in the cytosol and interact with docking and translocation subcomplexes on the peroxisomal membrane. Using immunoprecipitations of multiple protein components, we show that in Pichia pastoris the docking subcomplex consists of the unique peroxins Pex13p, Pex14p and Pex17p, whereas the putative translocation subcomplex has all three RING-finger peroxins, Pex2p, Pex10p and Pex12p, as unique constituents. We identify Pex3p as a shared component of both subcomplexes. In pex3Δ cells, the unique constituents of the docking subcomplex interact as they do in wild-type cells, but the assembly of the translocation subcomplex is impaired and its components are present at reduced levels. Furthermore, several interactions detected in wild-type cells between translocation and docking subcomplex components are undetectable in pex3Δ cells. Contrary to previous reports, pex3Δ cells have peroxisome remnants that pellet during high-speed centrifugation, associate with membranes on floatation gradients and can be visualized by deconvolution microscopy using antibodies to several peroxins which were not available earlier. We discuss roles for Pex3p in the assembly of specific peroxisomal membrane protein subcomplexes whose formation is necessary for matrix protein import.     

Pex2 and Pex12 Function as Protein-Ubiquitin Ligases in Peroxisomal Protein Import

The PTS1-dependent peroxisomal matrix protein import is facilitated by the receptor protein Pex5 and can be divided into cargo recognition in the cytosol, membrane docking of the cargo-receptor complex, cargo release, and recycling of the receptor. The final step is controlled by the ubiquitination status of Pex5. While polyubiquitinated Pex5 is degraded by the proteasome, monoubiquitinated Pex5 is destined for a new round of the receptor cycle. Recently, the ubiquitin-conjugating enzymes involved in Pex5 ubiquitination were identified as Ubc4 and Pex4 (Ubc10), whereas the identity of the corresponding protein-ubiquitin ligases remained unknown. Here we report on the identification of the protein-ubiquitin ligases that are responsible for the ubiquitination of the peroxisomal protein import receptor Pex5. It is demonstrated that each of the three RING peroxins Pex2, Pex10, and Pex12 exhibits ubiquitin-protein isopeptide ligase activity. Our results show that Pex2 mediates the Ubc4-dependent polyubiquitination whereas Pex12 facilitates the Pex4-dependent monoubiquitination of Pex5.The maintenance of peroxisome function depends on the formation of the peroxisomal membrane and the subsequent import of both membrane and matrix proteins. Without exception, peroxisomal matrix proteins are nucleus encoded, synthesized on free ribosomes, and subsequently imported in a posttranslational manner (40). The peroxisomal import apparatus can facilitate the transport of folded and oligomeric proteins over the peroxisomal membrane, with the basic principle of this translocation event still being unknown. Based on the concept of cycling receptors (9, 31), the receptor cycle is divided into four steps. In the first step, the cargo proteins are recognized in the cytosol by their cognate receptor protein Pex5 or Pex7. In general, this initial step depends on either one of the two well-characterized PTSs (peroxisomal targeting signals), PTS1 and PTS2, which are recognized and bound by the corresponding receptor proteins Pex5 and Pex7, respectively. In the second step, the cargo-loaded receptors dock with distinct proteins accessible at the surface of the peroxisomal membrane, namely, Pex13 and Pex14. These two proteins together with Pex17 are established components of the docking complex. A second complex of the peroxisomal protein import machinery acts downstream of the docking event and consists of the three peroxins Pex2, Pex10, and Pex12. A common feature of these proteins is a C-terminal RING (really interesting new gene) finger domain. The RING finger subcomplex and the docking subcomplex are both linked in a Pex8-dependent manner to form a larger assembly, the importomer (1). In the third step of the receptor cycle, the cargo is delivered to the peroxisomal matrix, and finally, the receptor is released from the peroxisomal membrane in an ATP-dependent manner and thus made available for proteasomal degradation or another round of import (for a review, see reference 27).With respect to the PTS1 receptor Pex5, recent reports demonstrated that this final ATP-dependent step in the receptor cycle is catalyzed by the AAA (ATPases associated with various cellular activities) peroxins Pex1 and Pex6 (33, 37). The signal for the export process is the attachment of a monoubiquitin moiety or, alternatively, the anchoring of a polyubiquitin chain (5, 35). This protein modification is in general facilitated by a three-step enzyme cascade (20). The ubiquitin (Ub)-activating enzyme (E1) activates the Ub and transfers it to the Ub conjugation enzyme (E2). In a final step, a protein-Ub ligase (E3) binds both E2 and substrate and thereby facilitates the conjugation of the Ub moiety onto the substrate protein. Saccharomyces cerevisiae harbors genes coding for one E1 enzyme, 11 E2 enzymes, and approximately 80 to 100 putative E3 enzymes (18, 29). It was demonstrated that the polyubiquitination of Pex5 primarily depends on the E2 protein Ubc4, which upon deletion can be partly replaced by Ubc5 or Ubc1 (22, 25, 36). Polyubiquitination of Pex5 is not a prerequisite for its function in peroxisomal protein import but might be a crucial step of a quality control system for the disposal of dysfunctional Pex5 (10, 22, 25, 36). Pex5 monoubiquitination is facilitated by the E2 protein Pex4 (Ubc10) in yeast or the Pex4-like UbcH5a/b/c in humans (14, 35, 47). The modification of Pex5 by a single Ub primes the receptor for its export back to the cytosol, where the Ub supposedly is removed prior to the initiation of a new receptor cycle (5, 14, 35). Although the functional relevance and the cognate E2 protein required for the different Ub modifications of Pex5 were identified, the factor(s) determining the substrate specificity, the protein-Ub ligase(s), remained unknown.Here we report on the discovery of the function of Pex2 and Pex12 as E3 proteins required for ubiquitination of the import receptor Pex5. These RING peroxins, defects of which cause the lethal peroxisome biogenesis disorders in humans, exhibit Ub-protein isopeptide ligase activity with Pex5 as the molecular target. Pex2 is shown to mediate the Ubc4-dependent polyubiquitination whereas Pex12 facilitates the Pex4-dependent monoubiquitination of Pex5.     

Two independent pathways traffic the intraperoxisomal peroxin PpPex8p into peroxisomes: mechanism and evolutionary implications

Among peroxins involved in peroxisome biogenesis, only Pex8p is predominantly intraperoxisomal at steady state. Pex8p is necessary for peroxisomal matrix protein import via the PTS1 and PTS2 pathways. It is proposed to bridge two peroxisomal membrane subcomplexes comprised of the docking (Pex13p, Pex14p, Pex17p) and RING (Pex2p, Pex10p, Pex12p) peroxins and is also implicated in cargo release of PTS1 proteins in the matrix. We show that Pichia pastoris Pex8p (PpPex8p) enters the peroxisome matrix using two redundant pathways in a Pex14p-dependent, but Pex2p-independent, manner, showing that the intact importomer and RING subcomplex are not required for its import. One pathway depends on the TPR motifs in Pex5p, the C-terminal PTS1 sequence (AKL) in PpPex8p, and the intraperoxisomal presence of this peroxin. The alternative pathway uses the PTS2 receptor, Pex7p, its accessory protein, Pex20p, and a putative PTS2 motif in PpPex8p, but does not require intraperoxisomal PpPex8p. Pex20p interaction with PpPex8p is independent of Pex7p, but the interaction of PpPex8p with Pex7p requires Pex20p. These data suggest a direct interaction between PpPex8p and Pex20p. Our studies shed light on the mechanism and evolution of the dual import pathways for PpPex8p.     

Pex10p functions as an E3 ligase for the Ubc4p-dependent ubiquitination of Pex5p

The Saccharomyces cerevisiae (Sc) PTS1 import receptor Pex5p is modified by ubiquitin, both in an Ubc4p-dependent and a Pex4p (Ubc10p)-dependent manner. Both of these modifications require the RING domain-containing protein Pex10p in vivo, but the actual role this protein plays in the ubiquitination of Pex5p has so far, remained enigmatic. Here, we report that the RING domain of Pex10p exhibits E₃ ligase activity in vitro, in combination with the human E₂ enzyme UbcH5a, a homologue of ScUbc4p, but not when ScPex4p was used as an E₂ enzyme in the reaction. We have further characterised Pex10p’s E₃ ligase activity using mutants designed to disturb this activity and show that Pex10p acts as the E₃ ligase for Ubc4p-dependent ubiquitination of Pex5p but not Pex4p-dependent ubiquitination in vivo. These data imply that the two distinct Pex5p modifications require different E₃ ligases, as well as different E₂ enzymes.     

The Cytosolic Domain of Pex22p Stimulates the Pex4p-Dependent Ubiquitination of the PTS1-Receptor

Peroxisomal biogenesis is an ubiquitin-dependent process because the receptors required for the import of peroxisomal matrix proteins are controlled via their ubiquitination status. A key step is the monoubiquitination of the import receptor Pex5p by the ubiquitin-conjugating enzyme (E2) Pex4p. This monoubiquitination is supposed to take place after Pex5p has released the cargo into the peroxisomal matrix and primes Pex5p for the extraction from the membrane by the mechano-enzymes Pex1p/Pex6p. These two AAA-type ATPases export Pex5p back to the cytosol for further rounds of matrix protein import. Recently, it has been reported that the soluble Pex4p requires the interaction to its peroxisomal membrane-anchor Pex22p to display full activity. Here we demonstrate that the soluble C-terminal domain of Pex22p harbours its biological activity and that this activity is independent from its function as membrane-anchor of Pex4p. We show that Pex4p can be functionally fused to the trans-membrane segment of the membrane protein Pex3p, which is not directly involved in Pex5p-ubiquitination and matrix protein import. However, this Pex3(N)-Pex4p chimera can only complement the double-deletion strain pex4Δ/pex22Δ and ensure optimal Pex5p-ubiquitination when the C-terminal part of Pex22p is additionally expressed in the cell. Thus, while the membrane-bound portion Pex22(N)p is not required when Pex4p is fused to Pex3(N)p, the soluble Pex22(C)p is essential for peroxisomal biogenesis and efficient monoubiquitination of the import receptor Pex5p by the E3-ligase Pex12p in vivo and in vitro. The results merge into a picture of an ubiquitin-conjugating complex at the peroxisomal membrane consisting of three domains: the ubiquitin-conjugating domain (Pex4p), a membrane-anchor domain (Pex22(N)p) and an enhancing domain (Pex22(C)p), with the membrane-anchor domain being mutually exchangeable, while the Ubc- and enhancer-domains are essential.     

The AAA-type ATPases Pex1p and Pex6p and their role in peroxisomal matrix protein import in Saccharomyces cerevisiae

The recognition of the conserved ATP-binding domains of Pex1p, p97 and NSF led to the discovery of the family of AAA-type ATPases. The biogenesis of peroxisomes critically depends on the function of two AAA-type ATPases, namely Pex1p and Pex6p, which provide the energy for import of peroxisomal matrix proteins. Peroxisomal matrix proteins are synthesized on free ribosomes in the cytosol and guided to the peroxisomal membrane by specific soluble receptors. At the membrane, the cargo-loaded receptors bind to a docking complex and the receptor-docking complex assembly is thought to form a dynamic pore which enables the transition of the cargo into the organellar lumen. The import cycle is completed by ubiquitination- and ATP-dependent dislocation of the receptor from the membrane to the cytosol, which is performed by the AAA-peroxins. Receptor ubiquitination and dislocation are the only energy-dependent steps in peroxisomal protein import. The export-driven import model suggests that the AAA-peroxins might function as motor proteins in peroxisomal import by coupling ATP-dependent removal of the peroxisomal import receptor and cargo translocation into the organelle.     

New insights into dynamic and functional assembly of the AAA peroxins, Pex1p and Pex6p, and their membrane receptor Pex26p in shuttling of PTS1-receptor Pex5p during peroxisome biogenesis

Peroxisome is a single-membrane organelle in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient peroxisome biogenesis disorders such as Zellweger syndrome. Two AAA peroxins, Pex1p and Pex6p, are encoded by PEX1 and PEX6, the causal genes for PBDs of complementation groups 1 and 4, respectively. PEX26 responsible for peroxisome biogenesis disorders of complementation group 8 codes for C-tail-anchored type-II membrane peroxin Pex26p, the recruiter of Pex1p-Pex6p complexes to peroxisomes. Pex1p is targeted to peroxisomes in a manner dependent on ATP hydrolysis, while Pex6p targeting requires ATP but not its hydrolysis. Pex1p and Pex6p are most likely regulated in their peroxisomal localization onto Pex26p via conformational changes by ATPase cycle. Pex5p is the cytosolic receptor for peroxisome matrix proteins with peroxisome targeting signal type-1 and shuttles between the cytosol and peroxisomes. AAA peroxins are involved in the export from peroxisomes of Pex5p. Pex5p is ubiquitinated at the conserved cysteine11 in a form associated with peroxisomes. Pex5p with a mutation of the cysteine11 to alanine, termed Pex5p-C11A, abrogates peroxisomal import of proteins harboring peroxisome targeting signals 1 and 2 in wild-type cells. Pex5p-C11A is imported into peroxisomes but not exported, hence suggesting an essential role of the cysteine residue in the export of Pex5p.     

The Hansenula polymorpha peroxisomal targeting signal 1 receptor, Pex5p, functions as a tetramer

We have studied Hansenula polymorpha Pex5p and Pex20p, peroxins involved in peroxisomal matrix protein import. In vitro binding experiments suggested that H. polymorpha Pex5p and Pex20p physically interact. We used single particle electron microscopy (EM) to analyze the structure of purified Pex5p and its possible association with Pex20p. Upon addition of Pex20p, a multimeric Pex20p complex was observed to be associated to the periphery of the Pex5p tetramer. In this Pex5p-Pex20p complex, the conformation of tetrameric Pex5p had changed from a closed conformation with a diameter of 115A into an open conformation of 134A. EM also indicated that the Pex5p-Pex20p complex was capable to bind native, folded catalase, a peroxisomal PTS1 protein. This suggests that the Pex5p-Pex20p complex may be functional as receptor complex.     

Mammalian Pex14p: membrane topology and characterisation of the Pex14p-Pex14p interaction

Peroxisomal biogenesis is a complex process requiring the action of numerous peroxins. One central component of this machinery is Pex14p, an intrinsic peroxisomal membrane protein probably involved in the docking of Pex5p, the receptor for PTS1-containing proteins (peroxisomal targeting signal 1-containing proteins). In this work the membrane topology of mammalian Pex14p was studied. Using a combination of protease protection assays and CNBr cleavage, we show that the first 130 amino acid residues of Pex14p are highly protected from exogenously added proteases by the peroxisomal membrane itself. Data indicating that this domain is responsible for the strong interaction of Pex14p with the organelle membrane are presented. All the other Pex14p amino acid residues are exposed to the cytosol. The properties of recombinant human Pex14p were also characterised. Heterologous expressed Pex14p was found to be a homopolymer of variable stoichiometry. Finally, in vitro binding assays indicate that homopolymerisation of Pex14p involves a domain comprising amino acid residues 147-278 of this peroxin.     

ATP-dependent assembly of the heteromeric Pex1p-Pex6p-complex of the peroxisomal matrix protein import machinery

The peroxisomal matrix protein import is facilitated by soluble receptor molecules which cycle between cytosol and the peroxisomal membrane. At the end of the receptor cycle, the import receptors are exported back to the cytosol in an ATP-dependent manner catalyzed by Pex1p and Pex6p, two AAA (ATPases associated with various cellular activities) type ATPases. Pex1p and Pex6p interact and form a heteromeric complex. In order to gain more insight into the stoichiometry and mechanism of assembly of the complex, we heterologously expressed and purified Saccharomyces cerevisiae Pex1p and Pex6p. Size exclusion chromatography studies of the recombinant proteins demonstrate that they form a hexameric complex in a one-to-one ratio of both AAA-proteins. The recombinant AAA-complex exhibits an ATPase activity with a k(m) of 0.17mM and V(max) of 0.35nmolmin(-1)μg(-1). In the presence of N-ethylmaleimide, ATPase activity of the peroxisomal AAA-complex is drastically decreased and the complex dissociates. Disassembly of the complex into its Pex1p and Pex6p subunits is also observed upon ATP-depletion, indicating that formation of the Pex1p/Pex6p-complex requires the presence of ATP.     

Functional association of the AAA complex and the peroxisomal importomer

The AAA peroxins, Pex1p and Pex6p, are components of the peroxisomal protein import machinery required for the relocation of the import receptor Pex5p from the peroxisomal membrane to the cytosol. We demonstrate that Pex1p and Pex6p form a stable complex in the cytosol, which associates at the peroxisomal membrane with their membrane anchor Pex15p and the peroxisomal importomer. The interconnection of Pex15p with the components of the importomer was independent of Pex1p and Pex6p, indicating that Pex15p is an incorporated component of the assembly. Further evidence suggests that the AAA peroxins shuttle between cytosol and peroxisome with proper binding of the Pex15p-AAA complex to the importomer and release of the AAA peroxins from the peroxisomal membrane depending on an operative peroxisomal protein import mechanism. Pex4p-deficient cells exhibit a wild-type-like assembly of the importomer, which differs in that it is associated with increased amounts of Pex1p and Pex6p, in agreement with a function for Pex4p in the release of AAA peroxins from the peroxisomal membrane.