Functional similarity between the peroxisomal PTS2 receptor binding protein Pex18p and the N-terminal half of the PTS1 receptor Pex5p

Within the extended receptor cycle of peroxisomal matrix import, the function of the import receptor Pex5p comprises cargo recognition and transport. While the C-terminal half (Pex5p-C) is responsible for PTS1 binding, the contribution of the N-terminal half of Pex5p (Pex5p-N) to the receptor cycle has been less clear. Here we demonstrate, using different techniques, that in Saccharomyces cerevisiae Pex5p-N alone facilitates the import of the major matrix protein Fox1p. This finding suggests that Pex5p-N is sufficient for receptor docking and cargo transport into peroxisomes. Moreover, we found that Pex5p-N can be functionally replaced by Pex18p, one of two auxiliary proteins of the PTS2 import pathway. A chimeric protein consisting of Pex18p (without its Pex7p binding site) fused to Pex5p-C is able to partially restore PTS1 protein import in a PEX5 deletion strain. On the basis of these results, we propose that the auxiliary proteins of the PTS2 import pathway fulfill roles similar to those of the N-terminal half of Pex5p in the PTS1 import pathway.     

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.     

Cysteine ubiquitination of PTS1 receptor Pex5p regulates Pex5p recycling

Pex5p is the cytosolic receptor for peroxisome matrix proteins with peroxisome-targeting signal (PTS) type 1 and shuttles between the cytosol and peroxisomes. Here, we show that Pex5p is ubiquitinated at the conserved cysteine(11) in a manner sensitive to dithiothreitol, in a form associated with peroxisomes. Pex5p with a mutation of the cysteine(11) to alanine, termed Pex5p-C11A, abrogates peroxisomal import of PTS1 and PTS2 proteins in wild-type cells. Pex5p-C11A is imported into peroxisomes but not exported, resulting in its accumulation in peroxisomes. These results suggest an essential role of the cysteine residue in the export of Pex5p. Furthermore, domain mapping indicates that N-terminal 158-amino-acid region of Pex5p-C11A, termed 158-CA, is sufficient for such dominant-negative activity by binding to membrane peroxin Pex14p via its two pentapeptide WXXXF/Y motifs. Stable expression of either Pex5p-C11A or 158-CA likewise inhibits the wild-type Pex5p import into peroxisomes, strongly suggesting that Pex5p-C11A exerts the dominant-negative effect at the translocation step via Pex14p. Taken together, these findings show that the cysteine(11) of Pex5p is indispensable for two distinct steps, its import and export. The Pex5p-C11A would be a useful tool for gaining a mechanistic insight into the matrix protein import into peroxisomes.     

The human peroxisomal targeting signal receptor, Pex5p, is translocated into the peroxisomal matrix and recycled to the cytosol

Peroxisomal targeting signals (PTSs) are recognized by predominantly cytosolic receptors, Pex5p and Pex7p. The fate of these PTS receptors following their interactions on the peroxisomal membrane with components of docking and putative translocation complexes is unknown. Using both novel and multiple experimental approaches, we show that human Pex5p does not just bind cargo and deliver it to the peroxisome membrane, but participates in multiple rounds of entry into the peroxisome matrix and export to the cytosol independent of the PTS2 import pathway. This unusual shuttling mechanism for the PTS1 receptor distinguishes protein import into peroxisomes from that into most other organelles, with the exception of the nucleus.     

The cytosolic peroxisome-targeting signal (PTS)-receptors,Pex7p and Pex5pL,are sufficient to transport PTS2 proteins to peroxisomes

Proteins harboring peroxisome-targeting signal type-2 (PTS2) are recognized in the cytosol by mobile PTS2 receptor Pex7p and associate with a longer isoform Pex5pL of the PTS1 receptor. Trimeric PTS2 protein-Pex7p-Pex5pL complexes are translocated to peroxisomes in mammalian cells. However, it remains unclear whether Pex5pL and Pex7p are sufficient cytosolic components in transporting of PTS2 proteins to peroxisomes. Here, we construct a semi-intact cell import system to define the cytosolic components required for the peroxisomal PTS2 protein import and show that the PTS2 pre-import complexes comprising Pex7p, Pex5p, and Hsc70 isolated from the cytosol of pex14 Chinese hamster ovary cell mutant ZP161 is import-competent. PTS2 reporter proteins are transported to peroxisomes by recombinant Pex7p and Pex5pL in semi-intact cells devoid of the cytosol. Furthermore, PTS2 proteins are translocated to peroxisomes in the presence of a non-hydrolyzable ATP analogue, adenylyl imidodiphosphate, and N-ethylmaleimide, suggesting that ATP-dependent chaperones including Hsc70 are dispensable for PTS2 protein import. Taken together, we suggest that Pex7p and Pex5pL are the minimal cytosolic factors in the transport of PTS2 proteins to peroxisomes.     

AWP1/ZFAND6 functions in Pex5 export by interacting with cys-monoubiquitinated Pex5 and Pex6 AAA ATPase

During biogenesis of the peroxisome, a subcellular organelle, the peroxisomal-targeting signal 1 (PTS1) receptor Pex5 functions as a shuttling receptor for PTS1-containing peroxisomal matrix proteins. However, the precise mechanism of receptor shuttling between peroxisomes and cytosol remains elusive despite the identification of numerous peroxins involved in this process. Herein, a new factor was isolated by a combination of biochemical fractionation and an in vitro Pex5 export assay, and was identified as AWP1/ZFAND6, a ubiquitin-binding NF-κB modulator. In the in vitro Pex5 export assay, recombinant AWP1 stimulated Pex5 export and an anti-AWP1 antibody interfered with Pex5 export. AWP1 interacted with Pex6 AAA ATPase, but not with Pex1-Pex6 complexes. Preferential binding of AWP1 to the cysteine-ubiquitinated form of Pex5 rather than to unmodified Pex5 was mediated by the AWP1 A20 zinc-finger domain. Inhibition of AWP1 by RNA interference had a significant effect on PTS1-protein import into peroxisomes. Furthermore, in AWP1 knock-down cells, Pex5 stability was decreased, similar to fibroblasts from patients defective in Pex1, Pex6 and Pex26, all of which are required for Pex5 export. Taken together, these results identify AWP1 as a novel cofactor of Pex6 involved in the regulation of Pex5 export during peroxisome biogenesis.     

Molecular mechanisms of import of peroxisome-targeting signal type 2 (PTS2) proteins by PTS2 receptor Pex7p and PTS1 receptor Pex5pL

In the present study, we investigated molecular mechanisms underlying the import of peroxisome-targeting signal type 2 (PTS2) proteins into peroxisomes. Purified Chinese hamster Pex7p that had been expressed in an Sf9/baculovirus system was biologically active in several assays such as those for PTS2 binding and assessing the restoration of the impaired PTS2 protein import in Chinese hamster ovary (CHO) pex7 mutant ZPG207. Pex7p was eluted as a monomer in gel filtration chromatography. Moreover, the mutation of the highly conserved cysteine residue suggested to be involved in the dimer formation did not affect the complementing activity in ZPG207 cells. Together, Pex7p more likely functions as a monomer. Together with PTS1 protein, the Pex7p-PTS2 protein complex was bound to Pex5pL, the longer form of Pex5p, which was prerequisite for the translocation of Pex7p-PTS2 protein complexes. Pex5pL-(Pex7p-PTS2 protein) complexes were detectable in wild-type CHO-K1 cells and were apparently more stable in pex14 CHO cells deficient in the entry site of the matrix proteins, whereas only the Pex7p-PTS2 protein complex was discernible in a Pex5pL-defective pex5 CHO mutant. Pex7p-PTS2 proteins bound to Pex14p via Pex5pL. In contrast, PTS2 protein-bound Pex7p as well as Pex7p directly and equally interacted with Pex13p, implying that the PTS2 cargo may be released at Pex13p. Furthermore, we detected the Pex13p complexes likewise formed with Pex5pL-bound Pex7p-PTS2 proteins. Thus, the Pex7p-mediated PTS2 protein import shares most of the steps with the Pex5p-dependent PTS1 import machinery but is likely distinct at the cargo-releasing stage.     

Pex20p functions as the receptor for non‐PTS1/non‐PTS2 acyl‐CoA oxidase import into peroxisomes of the yeast Yarrowia lipolytica

Most soluble proteins targeted to the peroxisomal matrix contain a C‐terminal peroxisome targeting signal type 1 (PTS1) or an N‐terminal PTS2 that is recognized by the receptors Pex5p and Pex7p, respectively. These receptors cycle between the cytosol and peroxisome and back again for multiple rounds of cargo delivery to the peroxisome. A small number of peroxisomal matrix proteins, including all six isozymes of peroxisomal fatty acyl‐CoA oxidase (Aox) of the yeast Yarrowia lipolytica, contain neither a PTS1 nor a PTS2. Pex20p has been shown to function as a co‐receptor for Pex7p in the import of PTS2 cargo into peroxisomes. Here we show that cells of Y. lipolytica deleted for the PEX20 gene fail to import not only the PTS2‐containing protein 3‐ketoacyl‐CoA thiolase (Pot1p) but also the non‐PTS1/non‐PTS2 Aox isozymes. Pex20p binds directly to Aox isozymes Aox3p and Aox5p, which requires the C‐terminal Wxxx(F/Y) motif of Pex20p. A W411G mutation in the C‐terminal Wxxx(F/Y) motif causes Aox isozymes to be mislocalized to the cytosol. Pex20p interacts physically with members of the peroxisomal import docking complex, Pex13p and Pex14p. Our results are consistent with a role for Pex20p as the receptor for import of the non‐PTS1/non‐PTS2 Aox isozymes into peroxisomes.     

PTS1-independent targeting of isocitrate lyase to peroxisomes requires the PTS1 receptor Pex5p

The targeting of castor bean isocitrate lyase to peroxisomes was studied by expression in the heterologous host Saccharomyces cerevisae from which the endogenous ICL1 gene had been removed by gene disruption. Peroxisomal import of ICL was dependent upon the PTS1 receptor Pex5p and was lost by deletion of the last three amino acids, Ala-Arg-Met. However, removal of an additional 16 amino acids restored the ability of this truncated ICL to be targeted to peroxisomes and this import activity, like that of the full-length protein, was dependent upon Pex5p. The ability of peptides corresponding to the carboxyl terminal ends of wild-type and Δ3 and Δ19 mutants of ICL to interact with the PTS1-binding portion of Pex5p from humans, plants and yeast was determined using the yeast two-hybrid system. The peptide corresponding to wild-type ICL interacted with all three Pex5p proteins to differing extents, but neither mutant could interact with Pex5p from any species. Thus, ICL can be targeted to peroxisomes in a Pex5p-dependent but PTS1-independent fashion. These results help to clarify the contradictory published data about the requirement of the PTS1 signal for ICL targeting.     

Pex18p and Pex21p,a Novel Pair of Related Peroxins Essential for Peroxisomal Targeting by the PTS2 Pathway

We have identified ScPex18p and ScPex21p, two novel S. cerevisiae peroxins required for protein targeting via the PTS2 branch of peroxisomal biogenesis. Targeting by this pathway is known to involve the interaction of oligopeptide PTS2 signals with Pex7p, the PTS2 receptor. Pex7p function is conserved between yeasts and humans, with defects in the human protein causing rhizomelic chondrodysplasia punctata (RCDP), a severe, lethal peroxisome biogenesis disorder characterized by aberrant targeting of several PTS2 peroxisomal proteins, but uncertainty remains about the subcellular localization of this receptor. Previously, we have reported that ScPex7p resides predominantly in the peroxisomal matrix, suggesting that it may function as a highly unusual intraorganellar import receptor, and the data presented in this paper identify Pex18p and Pex21p as key components in the targeting of Pex7p to peroxisomes. They each interact specifically with Pex7p both in two-hybrid analyses and in vitro. In cells lacking both Pex18p and Pex21p, Pex7p remains cytosolic and PTS2 targeting is completely abolished. Pex18p and Pex21p are weakly homologous to each other and display partial functional redundancy, indicating that they constitute a two-member peroxin family specifically required for Pex7p and PTS2 targeting.     

Yarrowia lipolytica Pex20p, Saccharomyces cerevisiae Pex18p/Pex21p and mammalian Pex5pL fulfil a common function in the early steps of the peroxisomal PTS2 import pathway

Import of peroxisomal matrix proteins is essential for peroxisome biogenesis. Genetic and biochemical studies using a variety of different model systems have led to the discovery of 23 PEX genes required for this process. Although it is generally believed that, in contrast to mitochondria and chloroplasts, translocation of proteins into peroxisomes involves a receptor cycle, there are reported differences of an evolutionary conservation of this cycle either with respect to the components or the steps involved in different organisms. We show here that the early steps of protein import into peroxisomes exhibit a greater similarity than was thought previously to be the case. Pex20p of Yarrowia lipolytica, Pex18p and Pex21p of Saccharomyces cerevisiae and mammalian Pex5pL fulfil a common function in the PTS2 pathway of their respective organisms. These non-orthologous proteins possess a conserved sequence region that most likely represents a common PTS2-receptor binding site and di-aromatic pentapeptide motifs that could be involved in binding of the putative docking proteins. We propose that not necessarily the same proteins but functional modules of them are conserved in the early steps of peroxisomal protein import.     

The Peroxisomal Matrix Import of Pex8p Requires Only PTS Receptors and Pex14p

Pichia pastoris (Pp) Pex8p, the only known intraperoxisomal peroxin at steady state, is targeted to peroxisomes by either the peroxisomal targeting signal (PTS) type 1 or PTS2 pathway. Until recently, all cargoes entering the peroxisome matrix were believed to require the docking and really interesting new gene (RING) subcomplexes, proteins that bridge these two subcomplexes and the PTS receptor-recycling machinery. However, we reported recently that the import of PpPex8p into peroxisomes via the PTS2 pathway is Pex14p dependent but independent of the RING subcomplex (Zhang et al., 2006 ). In further characterizing the peroxisome membrane-associated translocon, we show that two other components of the docking subcomplex, Pex13p and Pex17p, are dispensable for the import of Pex8p. Moreover, we demonstrate that the import of Pex8p via the PTS1 pathway also does not require the RING subcomplex or intraperoxisomal Pex8p. In receptor-recycling mutants (Δpex1, Δpex6, and Δpex4), Pex8p is largely cytosolic because Pex5p and Pex20p are unstable. However, upon overexpression of the degradation-resistant Pex20p mutant, hemagglutinin (HA)-Pex20p(K19R), in Δpex4 and Δpex6 cells, Pex8p enters peroxisome remnants. Our data support the idea that PpPex8p is a special cargo whose translocation into peroxisomes depends only on the PTS receptors and Pex14p and not on intraperoxisomal Pex8p, the RING subcomplex, or the receptor-recycling machinery.     

PTS1-independent targeting of isocitrate lyase to peroxisomes requires the PTS1 receptor Pex5p

The targeting of castor bean isocitrate lyase to peroxisomes was studied by expression in the heterologous host Saccharomyces cerevisae from which the endogenous ICL1 gene had been removed by gene disruption. Peroxisomal import of ICL was dependent upon the PTS1 receptor Pex5p and was lost by deletion of the last three amino acids, Ala-Arg-Met. However, removal of an additional 16 amino acids restored the ability of this truncated ICL to be targeted to peroxisomes and this import activity, like that of the full-length protein, was dependent upon Pex5p. The ability of peptides corresponding to the carboxyl terminal ends of wild-type and Delta 3 and Delta 19 mutants of ICL to interact with the PTS1-binding portion of Pex5p from humans, plants and yeast was determined using the yeast two-hybrid system. The peptide corresponding to wild-type ICL interacted with all three Pex5p proteins to differing extents, but neither mutant could interact with Pex5p from any species. Thus, ICL can be targeted to peroxisomes in a Pex5p-dependent but PTS1-independent fashion. These results help to clarify the contradictory published data about the requirement of the PTS1 signal for ICL targeting.     

Multiple PEX genes are required for proper subcellular distribution and stability of Pex5p, the PTS1 receptor: evidence that PTS1 protein import is mediated by a cycling receptor

PEX5 encodes the type-1 peroxisomal targeting signal (PTS1) receptor, one of at least 15 peroxins required for peroxisome biogenesis. Pex5p has a bimodal distribution within the cell, mostly cytosolic with a small amount bound to peroxisomes. This distribution indicates that Pex5p may function as a cycling receptor, a mode of action likely to require interaction with additional peroxins. Loss of peroxins required for protein translocation into the peroxisome (PEX2 or PEX12) resulted in accumulation of Pex5p at docking sites on the peroxisome surface. Pex5p also accumulated on peroxisomes in normal cells under conditions which inhibit protein translocation into peroxisomes (low temperature or ATP depletion), returned to the cytoplasm when translocation was restored, and reaccumulated on peroxisomes when translocation was again inhibited. Translocation inhibiting conditions did not result in Pex5p redistribution in cells that lack detectable peroxisomes. Thus, it appears that Pex5p can cycle repeatedly between the cytoplasm and peroxisome. Altered activity of the peroxin defective in CG7 cells leads to accumulation of Pex5p within the peroxisome, indicating that Pex5p may actually enter the peroxisome lumen at one point in its cycle. In addition, we found that the PTS1 receptor was extremely unstable in the peroxin-deficient CG1, CG4, and CG8 cells. Altered distribution or stability of the PTS1 receptor in all cells with a defect in PTS1 protein import implies that the genes mutated in these cell lines encode proteins with a direct role in peroxisomal protein import.     

Identification of Pex13p a peroxisomal membrane receptor for the PTS1 recognition factor

We have identified an S. cerevisiae integral peroxisomal membrane protein of M of 42,705 (Pex13p) that is a component of the peroxisomal protein import apparatus. Pex13p's most striking feature is an src homology 3 (SH3) domain that interacts directly with yeast Pex5p (former Pas10p), the recognition factor for the COOH-terminal tripeptide signal sequence (PTS1), but not with Pex7p (former Pas7p), the recognition factor for the NH2-terminal nonapeptide signal (PTS2) of peroxisomal matrix proteins. Hence, Pex13p serves as peroxisomal membrane receptor for at least one of the two peroxisomal signal recognition factors. Cells deficient in Pex13p are unable to import peroxisomal matrix proteins containing PTS1 and, surprisingly, also those containing PTS2. Pex13p deficient cells retain membranes containing the peroxisomal membrane protein Pex11p (former Pmp27p), consistent with the existence of independent pathways for the integration of peroxisomal membrane proteins and for the translocation of peroxisomal matrix proteins.     

Peroxisomal Targeting of PTS2 Pre-Import Complexes in the Yeast Saccharomyces cerevisiae

Posttranslational matrix protein import into peroxisomes uses either one of the two peroxisomal targeting signals (PTS), PTS1 and PTS2. Unlike the PTS1 receptor Pex5p, the PTS2 receptor Pex7p is necessary but not sufficient to target cargo proteins into the peroxisomal matrix and requires coreceptors. Saccharomyces cerevisiae possesses two coreceptors, Pex18p and Pex21p, with a redundant but not a clearly defined function. To gain further insight into the early events of this import pathway, PTS2 pre-import complexes of S. cerevisiae were isolated and characterized by determination of size and protein composition in wild-type and different mutant strains. Mass spectrometric analysis of the cytosolic PTS2 pre-import complex indicates that Fox3p is the only abundant PTS2 protein under oleate growth conditions. Our data strongly suggest that the formation of the ternary cytosolic PTS2 pre-import complex occurs hierarchically. First, Pex7p recognizes cargo proteins through its PTS2 in the cytosol. In a second step, the coreceptor binds to this complex, and finally, this ternary 150 kDa pre-import complex docks at the peroxisomal membrane, where both the PTS1 and the PTS2 import pathways converge. Gel filtration analysis of membrane-bound subcomplexes suggests that Pex13p provides the initial binding partner at the peroxisomal membrane, whereas Pex14p assembles with Pex18p in high-molecular-weight complexes after or during dissociation of the PTS2 receptor.     

The deubiquitination of the PTS1-import receptor Pex5p is required for peroxisomal matrix protein import

Peroxisomal biogenesis depends on the correct import of matrix proteins into the lumen of the organelle. Most peroxisomal matrix proteins harbor the peroxisomal targeting-type 1 (PTS1), which is recognized by the soluble PTS1-receptor Pex5p in the cytosol. Pex5p ferries the PTS1-proteins to the peroxisomal membrane and releases them into the lumen. Finally, the PTS1-receptor is monoubiquitinated on the conserved cysteine 6 in Saccharomyces cerevisiae. The monoubiquitinated Pex5p is recognized by the peroxisomal export machinery and is retrotranslocated into the cytosol for further rounds of protein import. However, the functional relevance of deubiquitination has not yet been addressed.In this study, we have analyzed a Pex5p-truncation lacking Cys6 [(Δ6)Pex5p], a construct with a ubiquitin-moiety genetically fused to the truncation [Ub-(Δ6)Pex5p], as well as a construct with a reduced susceptibility to deubiquitination [Ub(G75/76A)-(Δ6)Pex5p]. While the (Δ6)Pex5p-truncation is not functional, the Ub-(Δ6)Pex5p chimeric protein can facilitate matrix protein import. In contrast, the Ub(G75/76A)-(Δ6)Pex5p chimera exhibits a complete PTS1-import defect. The data show for the first time that not only ubiquitination but also deubiquitination rates are tightly regulated and that efficient deubiquitination of Pex5p is essential for peroxisomal biogenesis.     

Saccharomyces cerevisiae acyl-CoA oxidase follows a novel, non-PTS1, import pathway into peroxisomes that is dependent on Pex5p

The peroxisomal protein acyl-CoA oxidase (Pox1p) of Saccharomyces cerevisiae lacks either of the two well characterized peroxisomal targeting sequences known as PTS1 and PTS2. Here we demonstrate that peroxisomal import of Pox1p is nevertheless dependent on binding to Pex5p, the PTS1 import receptor. The interaction between Pex5p and Pox1p, however, involves novel contact sites in both proteins. The interaction region in Pex5p is located in a defined area of the amino-terminal part of the protein outside of the tetratricopeptide repeat domain involved in PTS1 recognition; the interaction site in Pox1p is located internally and not at the carboxyl terminus where a PTS1 is normally found. By making use of pex5 mutants that are either specifically disturbed in binding of PTS1 proteins or in binding of Pox1p, we demonstrate the existence of two independent, Pex5p-mediated import pathways into peroxisomes in yeast as follows: a classical PTS1 pathway and a novel, non-PTS1 pathway for Pox1p.     

The SH3 domain of the Saccharomyces cerevisiae peroxisomal membrane protein Pex13p functions as a docking site for Pex5p, a mobile receptor for the import PTS1-containing proteins

We identified a Saccharomyces cerevisiae peroxisomal membrane protein, Pex13p, that is essential for protein import. A point mutation in the COOH-terminal Src homology 3 (SH3) domain of Pex13p inactivated the protein but did not affect its membrane targeting. A two-hybrid screen with the SH3 domain of Pex13p identified Pex5p, a receptor for proteins with a type I peroxisomal targeting signal (PTS1), as its ligand. Pex13p SH3 interacted specifically with Pex5p in vitro. We determined, furthermore, that Pex5p was mainly present in the cytosol and only a small fraction was associated with peroxisomes. We therefore propose that Pex13p is a component of the peroxisomal protein import machinery onto which the mobile Pex5p receptor docks for the delivery of the selected PTS1 protein.     

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.