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.     

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.     

Pex5p imports folded tetrameric catalase by interaction with pex13p

Human catalase forms a 240-kDa tetrameric complex and degrades H(2) O(2) in peroxisomes. Human catalase is targeted to peroxisomes by the interaction of its peroxisomal targeting signal type 1 (PTS1)-like KANL sequence with the cytosolic PTS1 receptor Pex5p. We show herein that human catalase tetramers are formed in the cytoplasm and that the expression of a PTS signal on each of the four subunits is not necessary for peroxisomal transport. We previously demonstrated that a Pex5p mutant defective in binding to Pex13p, designated Pex5p(Mut234), imports typical PTS1-type proteins but not catalase. This impaired catalase import is not rescued by replacing its C-terminal KANL sequence with a typical PTS1 sequence, SKL, indicating that the failure of catalase import in Mut234-expressing cells is not due to its weak PTS1. In contrast, several enzymatically inactive and monomeric mutants of catalase are efficiently imported in Mut234-expressing cells. Moreover, trimeric chloramphenicol acetyltransferase (CAT) harboring SKL is not imported in Pex5p(Mut234)-expressing cells, but CAT-SKL trimers are transported to peroxisomes in the wild-type cells. These findings suggest that the Pex5p-Pex13p interaction likely plays a pivotal role in the peroxisomal import of folded and oligomeric proteins.     

Peroxisomal membrane fusion requires two AAA family ATPases, Pex1p and Pex6p

Two AAA family ATPases, NSF and p97, have been implicated in membrane fusion during assembly and inheritance of organelles of the secretory pathway. We have now investigated the roles of AAA ATPases in membrane fusion during assembly of the peroxisome, an organelle outside the classical secretory system. Here, we show that peroxisomal membrane fusion in the yeast Yarrowia lipolytica requires two AAA ATPases, Pex1p and Pex6p. Release of membrane- associated Pex1p and Pex6p drives the asymmetric priming of two fusion partners. The next step, peroxisome docking, requires release of Pex1p from one partner. Subsequent fusion of the peroxisomal membranes is independent of both Pex1p and Pex6p.     

AAA Peroxins and Their Recruiter Pex26p Modulate the Interactions of Peroxins Involved in Peroxisomal Protein Import

Pex1p and Pex6p are required for the relocation of the import receptor Pex5p from the peroxisomal membrane to the cytosol. We herein show that mammalian Pex26p directly binds to Pex14p, the initial docking receptor of Pex5p, and interacts with Pex5p via Pex14p. The binding affinity of Pex26p to Pex14p is altered by Pex5p. Further evidence suggests that the N-terminal region in Pex26p acts as a scaffold protein to recruit Pex14p·Pex5p complex together with Pex1p·Pex6p complexes on peroxisomes. Pex26p binding to Pex14p was suppressed by overexpression of Pex1p and Pex6p in an ATP-dependent manner, whereas Pex14p was not competed out by Pex1p and Pex6p from Pex26p mutant defective in peroxisomal matrix protein import. These results suggested that peroxisome biogenesis requires Pex1p- and Pex6p-regulated dissociation of Pex14p from Pex26p. Pex1p homo-oligomer directly binds to Pex5p as assessed by a surface plasmon resonance-based assay. Moreover, cytosolic Pex1p is likely to maintain the functional oligomer of Pex5p. Taken together, in the peroxisomal protein import, AAA peroxins modulate the interaction between Pex26p and Pex14p on peroxisome membrane as well as Pex5p oligomer in the cytosol.     

Cysteine-specific ubiquitination protects the peroxisomal import receptor Pex5p against proteasomal degradation

Peroxisomal matrix protein import is mediated by dynamic import receptors, which cycle between the peroxisomal membrane and the cytosol. Proteins with a type 1 peroxisomal targeting signal (PTS1) are bound by the import receptor Pex5p in the cytosol and guided to the peroxisomal membrane. After cargo translocation into the peroxisomal matrix, the receptor is released from the membrane back to the cytosol in an ATP-dependent manner by the AAA-type ATPases Pex1p and Pex6p. These mechanoenzymes recognize ubiquitinated Pex5p-species as substrates for membrane extraction. The PTS1-receptor is either polyubiquitinated via peptide bonds at two certain lysines and results in proteasomal degradation or monoubiquitinated via a thioester-bond at a conserved cysteine, which enables the recycling of Pex5p and further rounds of matrix protein import. To investigate the physiological relevance of the conserved N-terminal cysteine of Pex5p, the known target amino acids for ubiquitination were substituted by site-directed mutagenesis. In contrast with Pex5p_C6A, Pex5p_C6K turned out to be functional in PTS1 import and utilization of oleic acid, independent of the lysines at position 18 and 24. In contrast with wild-type Pex5p, Pex5p_C6K displays an ubiquitination pattern, similar to the polyubiquitination pattern of Pex4p or Pex22p mutant strains. Moreover, Pex5p_C6K displays a significantly reduced steady-state level when the deubiquitinating enzyme Ubp15p is missing. Thus, our results indicate that not the cysteine residue but the position of ubiquitination is important for Pex5p function. The presence of the cysteine prevents polyubiquitination and rapid degradation of Pex5p.     

The peroxin Pex19p interacts with multiple, integral membrane proteins at the peroxisomal membrane

Pex19p is a protein required for the early stages of peroxisome biogenesis, but its precise function and site of action are unknown. We tested the interaction between Pex19p and all known Pichia pastoris Pex proteins by the yeast two-hybrid assay. Pex19p interacted with six of seven known integral peroxisomal membrane proteins (iPMPs), and these interactions were confirmed by coimmunoprecipitation. The interactions were not reduced upon inhibition of new protein synthesis, suggesting that they occur with preexisting, and not newly synthesized, pools of iPMPs. By mapping the domains in six iPMPs that interact with Pex19p and the iPMP sequences responsible for targeting to the peroxisome membrane (mPTSs), we found the majority of these sites do not overlap. Coimmunoprecipitation of Pex19p from fractions that contain peroxisomes or cytosol revealed that the interactions between predominantly cytosolic Pex19p and the iPMPs occur in the organelle pellet that contains peroxisomes. These data, taken together, suggest that Pex19p may have a chaperone-like role at the peroxisome membrane and that it is not the receptor for targeting of iPMPs to the peroxisome.     

Deficiency of the exportomer components Pex1, Pex6, and Pex15 causes enhanced pexophagy in Saccharomyces cerevisiae

Turnover of damaged, dysfunctional, or excess organelles is critical to cellular homeostasis. We screened mutants disturbed in peroxisomal protein import, and found that a deficiency in the exportomer subunits Pex1, Pex6, and Pex15 results in enhanced turnover of peroxisomal membrane structures compared with other mutants. Strikingly, almost all peroxisomal membranes were associated with phagophore assembly sites in pex1Δ atg1Δ cells. Degradation depended on Atg11 and the pexophagy receptor Atg36, which mediates degradation of superfluous peroxisomes. Mutants of PEX1, PEX6, and PEX15 accumulate ubiquitinated receptors at the peroxisomal membrane. This accumulation has been suggested to trigger pexophagy in mammalian cells. We show by genetic analysis that preventing this accumulation does not abolish pexophagy in Saccharomyces cerevisiae. We find Atg36 is modified in pex1Δ cells even when Atg11 binding is prevented, suggesting Atg36 modification is an early event in the degradation of dysfunctional peroxisomal structures in pex1Δ cells via pexophagy.     

Dynamic and functional assembly of the AAA peroxins, Pex1p and Pex6p, and their membrane receptor Pex26p

Two AAA peroxins, Pex1p and Pex6p, are encoded by PEX1 and PEX6, the causal genes for peroxisome biogenesis disorders of complementation group 1 (CG1) and CG4, respectively. PEX26 responsible for peroxisome biogenesis disorders of CG8 encodes Pex26p, the recruiter of Pex1p.Pex6p complexes to peroxisomes. We herein assigned the binding regions between human Pex1p and Pex6p and elucidated pivotal roles of the AAA cassettes, called D1 and D2 domains, in Pex1p-Pex6p interaction and peroxisome biogenesis. ATP binding in both AAA cassettes but not ATP hydrolysis in D2 of both Pex1p and Pex6p was prerequisite for Pex1p-Pex6p interaction and their peroxisomal localization. The AAA cassettes, D1 and D2, were essential for peroxisome-restoring activity of Pex1p and Pex6p. In HEK293 cells, endogenous Pex1p was partly localized likely as a homo-oligomer in the cytoplasm, while Pex6p and Pex26p were predominantly localized on peroxisomes. Interaction of Pex1p with Pex6p conferred a conformational change and dissociation of the Pex1p oligomer. These results suggested that Pex1p possesses two distinct oligomeric forms, a homo-oligomer in the cytosol and a hetero-oligomer on peroxisome membranes, possibly playing distinct functions in peroxisome biogenesis.     

Deficiency of the exportomer components Pex1, Pex6, and Pex15 causes enhanced pexophagy in Saccharomyces cerevisiae

Turnover of damaged, dysfunctional, or excess organelles is critical to cellular homeostasis. We screened mutants disturbed in peroxisomal protein import, and found that a deficiency in the exportomer subunits Pex1, Pex6, and Pex15 results in enhanced turnover of peroxisomal membrane structures compared with other mutants. Strikingly, almost all peroxisomal membranes were associated with phagophore assembly sites in pex1Δ atg1Δ cells. Degradation depended on Atg11 and the pexophagy receptor Atg36, which mediates degradation of superfluous peroxisomes. Mutants of PEX1, PEX6, and PEX15 accumulate ubiquitinated receptors at the peroxisomal membrane. This accumulation has been suggested to trigger pexophagy in mammalian cells. We show by genetic analysis that preventing this accumulation does not abolish pexophagy in Saccharomyces cerevisiae. We find Atg36 is modified in pex1Δ cells even when Atg11 binding is prevented, suggesting Atg36 modification is an early event in the degradation of dysfunctional peroxisomal structures in pex1Δ cells via pexophagy.     

The N-terminal half of the peroxisomal cycling receptor Pex5p is a natively unfolded domain

Targeting of most newly synthesised peroxisomal matrix proteins to the organelle requires Pex5p, the so-called PTS1 receptor. According to current models of peroxisomal biogenesis, Pex5p interacts with these proteins in the cytosol, transports them to the peroxisomal membrane and catalyses their translocation across the membrane. Presently, our knowledge on the structural details behind the interaction of Pex5p with the cargo proteins is reasonably complete. In contrast, information regarding the structure of the Pex5p N-terminal half (a region containing its peroxisomal targeting domain) is still limited. We have recently observed that the Stokes radius of this Pex5p domain is anomalously large, suggesting that this portion of the protein is either a structured elongated domain or that it adopts a low compactness conformation. Here, we address this issue using a combination of biophysical and biochemical approaches. Our results indicate that the N-terminal half of Pex5p is best described as a natively unfolded pre-molten globule-like domain. The implications of these findings on the mechanism of protein import into the peroxisome are discussed.     

Cysteine-dependent ubiquitination of Pex18p is linked to cargo translocation across the peroxisomal membrane

The peroxisomal matrix protein import is facilitated by cycling receptor molecules that shuttle between the cytosol and the peroxisomal membrane. In the yeast Saccharomyces cerevisiae, the import of proteins harboring a peroxisomal targeting signal of type II (PTS2) is mediated by the receptor Pex7p and its co-receptor Pex18p. Here we demonstrate that Pex18p undergoes two kinds of ubiquitin modifications. One of these ubiquitination events depends on lysines 13 and 20 and forces rapid Pex18p turnover by proteasomal degradation. A cysteine residue near the extreme Pex18p amino-terminus is required for the second type of ubiquitination. It turned out that this cysteine residue at position 6 is essential for the function of Pex18p in peroxisomal protein import but does not contribute to receptor-cargo association and binding to the peroxisomal import apparatus. However, in contrast to the wild-type protein, cysteine 6-mutated Pex18p is arrested in a membrane-protected state, whereas Pex7p is accessible in a protease protection assay. This finding indicates that Pex18p export is linked to cargo translocation, which supports the idea of an export-driven import of proteins into peroxisomes.     

Reassembly of peroxisomes in Hansenula polymorpha pex3 cells on reintroduction of Pex3p involves the nuclear envelope

The reassembly of peroxisomes in Hansenula polymorpha pex3 cells on reintroduction of Pex3p was examined. Using a Pex3-green fluorescent protein (Pex3-GFP) fusion protein, expressed under the control of an inducible promoter, it was observed that, initially on induction of Pex3-GFP synthesis, GFP fluorescence was localized to the endoplasmic reticulum and the nuclear envelope. Subsequently, a single organelle developed per cell that increased in size and multiplied by division. At these stages, GFP fluorescence was confined to peroxisomes. Fractionation experiments on homogenates of pex3 cells, in which the endoplasmic reticulum and nuclear envelope were marked with GFP, identified a small amount of GFP in peroxisomes present in the initial stage of peroxisome reassembly. Our data suggest a crucial role for the endoplasmic reticulum/nuclear envelope in peroxisome reintroduction on complementation of pex3 cells by the PEX3 gene.     

Role of Pex21p for Piggyback Import of Gpd1p and Pnc1p into Peroxisomes of Saccharomyces cerevisiae

Proteins designated for peroxisomal protein import harbor one of two common peroxisomal targeting signals (PTS). In the yeast Saccharomyces cerevisiae, the oleate-induced PTS2-dependent import of the thiolase Fox3p into peroxisomes is conducted by the soluble import receptor Pex7p in cooperation with the auxiliary Pex18p, one of two supposedly redundant PTS2 co-receptors. Here, we report on a novel function for the co-receptor Pex21p, which cannot be fulfilled by Pex18p. The data establish Pex21p as a general co-receptor in PTS2-dependent protein import, whereas Pex18p is especially important for oleate-induced import of PTS2 proteins. The glycerol-producing PTS2 protein glycerol-3-phosphate dehydrogenase Gpd1p shows a tripartite localization in peroxisomes, in the cytosol, and in the nucleus under osmotic stress conditions. We show the following: (i) Pex21p is required for peroxisomal import of Gpd1p as well as a key enzyme of the NAD⁺ salvage pathway, Pnc1p; (ii) Pnc1p, a nicotinamidase without functional PTS2, is co-imported into peroxisomes by piggyback transport via Gpd1p. Moreover, the specific transport of these two enzymes into peroxisomes suggests a novel regulatory role for peroxisomes under various stress conditions.     

In the yeast Hansenula polymorpha, peroxisome formation from the ER is independent of Pex19p, but involves the function of p24 proteins

The peroxin Pex19p is important for the formation of functional peroxisomal membranes. Here we show that Hansenula polymorpha Pex19p is also required for peroxisome inheritance. Peroxisome inheritance is partly defective when Pex19p farnesylation is blocked, whereas deletion of PEX19 resulted in a severe defect in partitioning of peroxisomal structures. Time lapse imaging revealed that in newly formed buds, which had not inherited a peroxisome from the mother cell, new peroxisomes are formed that derive from the nuclear envelope/endoplasmic reticulum. This process was impaired upon deletion of EMP24 and ERP3, genes that encode p24 proteins. p24 Proteins are components of coated vesicles that mediate trafficking between the endoplasmic reticulum and Golgi apparatus. In an H. polymorpha wild-type background, deletion of EMP24 and ERP3 resulted in a strong reduction of organelle number in conjunction with an increase in the size of individual peroxisomes. This observation suggests that p24 proteins also play a role in peroxisome development in wild-type H. polymorpha cells.     

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 peroxisomal AAA ATPase complex prevents pexophagy and development of peroxisome biogenesis disorders

Peroxisome biogenesis disorders (PBDs) are metabolic disorders caused by the loss of peroxisomes. The majority of PBDs result from mutation in one of 3 genes that encode for the peroxisomal AAA ATPase complex (AAA-complex) required for cycling PEX5 for peroxisomal matrix protein import. Mutations in these genes are thought to result in a defect in peroxisome assembly by preventing the import of matrix proteins. However, we show here that loss of the AAA-complex does not prevent matrix protein import, but instead causes an upregulation of peroxisome degradation by macroautophagy, or pexophagy. The loss of AAA-complex function in cells results in the accumulation of ubiquitinated PEX5 on the peroxisomal membrane that signals pexophagy. Inhibiting autophagy by genetic or pharmacological approaches rescues peroxisome number, protein import and function. Our findings suggest that the peroxisomal AAA-complex is required for peroxisome quality control, whereas its absence results in the selective degradation of the peroxisome. Thus the loss of peroxisomes in PBD patients with mutations in their peroxisomal AAA-complex is a result of increased pexophagy. Our study also provides a framework for the development of novel therapeutic treatments for PBDs.     

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.     

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.     

A conserved alpha helical domain at the N-terminus of Pex14p is required for PTS1 and PTS2 protein import in Hansenula polymorpha

We have analyzed the highly conserved N-terminus of Hansenula polymorpha Pex14p for its function in peroxisomal matrix protein import. The region comprising aa 10-54 of HpPex14p is predicted to contain three alpha-helices. Its alpha-helical structure was confirmed by CD analysis of a synthetic peptide, corresponding to residues 8-58. Deletion of aa 1-21 of HpPex14p, but not of aa 1-9, completely abolished PTS1 and PTS2 matrix protein import. An extensive mutational analysis of the first alpha-helix (aa 10-21) demonstrated that its secondary structure, as well as residues Phe20 and Leu21, are essential for PTS1 and PTS2 matrix protein import.