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.     

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.     

Redox-regulated Cargo Binding and Release by the Peroxisomal Targeting Signal Receptor,Pex5

In its role as a mobile receptor for peroxisomal matrix cargo containing a peroxisomal targeting signal called PTS1, the protein Pex5 shuttles between the cytosol and the peroxisome lumen. Pex5 binds PTS1 proteins in the cytosol via its C-terminal tetratricopeptide domains and delivers them to the peroxisome lumen, where the receptor·cargo complex dissociates. The cargo-free receptor is exported to the cytosol for another round of import. How cargo release and receptor recycling are regulated is poorly understood. We found that Pex5 functions as a dimer/oligomer and that its protein interactions with itself (homo-oligomeric) and with Pex8 (hetero-oligomeric) control the binding and release of cargo proteins. These interactions are controlled by a redox-sensitive amino acid, cysteine 10 of Pex5, which is essential for the formation of disulfide bond-linked Pex5 forms, for high affinity cargo binding, and for receptor recycling. Disulfide bond-linked Pex5 showed the highest affinity for PTS1 cargo. Upon reduction of the disulfide bond by dithiothreitol, Pex5 transitioned to a noncovalent dimer, concomitant with the partial release of PTS1 cargo. Additionally, dissipation of the redox balance between the cytosol and the peroxisome lumen caused an import defect. A hetero-oligomeric interaction between the N-terminal domain (amino acids 1–110) of Pex5 and a conserved motif at the C terminus of Pex8 further facilitates cargo release, but only under reducing conditions. This interaction is also important for the release of PTS1 proteins. We suggest a redox-regulated model for Pex5 function during the peroxisomal matrix protein import cycle.     

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.     

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.     

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.     

Saccharomyces cerevisiae Pex14p contains two independent Pex5p binding sites, which are both essential for PTS1 protein import

Pex14p is a peroxisomal membrane-associated protein involved in docking of both Pex5p and Pex7p to the peroxisomal membrane. Previous studies have shown that, in humans, the N-terminal region of Pex14p interacts with WxxxF/Y motifs in Pex5p. Here, we report that Saccharomyces cerevisiae Pex14p contains two independent Pex5p binding sites, one in the N- and one in the C-terminus. Using deletion analysis we show that, in vivo, both of these interactions are needed for PTS1 import. Furthermore, we show that the characterized WxxxF/Y motifs of Pex5p are not essential for binding to the N-terminus of Pex14p but do play a role in the interaction with the Pex14 C-terminus. Thus, the data suggest that the mechanism of the Pex14p-Pex5p interaction in yeast is different from that previously reported for humans.     

Distinct Ubiquitination Cascades Act on the Peroxisomal Targeting Signal Type 2 Co‐receptor Pex18p

Peroxisomal matrix protein import is facilitated by cycling receptors that recognize their cargo proteins in the cytosol by a peroxisomal targeting sequence (PTS) and ferry them to the peroxisomal membrane. Subsequently, the cargo is translocated into the peroxisomal lumen, whereas the receptor is released to the cytosol for further rounds of protein import. This cycle is controlled by the ubiquitination status of the receptor, which is best understood for the PTS1‐receptor. While polyubiquitination of PTS‐receptors results in their proteasomal degradation, the monoubiquitinated PTS‐receptors are exported to the cytosol and recycled for further rounds of protein import. Here, we describe the identification of two ubiquitination cascades acting on the PTS2 co‐receptor Pex18p. Using in vivo and in vitro approaches, we demonstrate that the polyubiquitination of Pex18p requires the ubiquitin‐conjugating enzyme (E2) Ubc4p, which cooperates with the RING (really interesting new gene)‐type ubiquitin‐protein ligases (E3) Pex2p as well as Pex10p. Monoubiquitination of Pex18p depends on the E2 enzyme Pex4p (Ubc10p), which functions in concert with the E3 enzymes Pex12p and Pex10p. Our findings for the PTS2‐pathway complement the data on PTS1‐receptor ubiquitination and add up to a unified concept of the ubiquitin‐based regulation of peroxisomal import .     

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.     

ARF- and coatomer-mediated peroxisomal vesiculation

The authors characterized on a molecular level the clofibrate-inducible 26-kDa integral peroxisomal membrane protein (Pmp26p, Pex11-1p) of rat liver. By screening cDNA databases with the obtained Pex11-1p-cDNA, a second homologous cDNA was identified that codes for a polypeptide with slightly larger molecular mass than Pex11-1p. The authors call this polypeptide Pex11-2p. Studies on the topology of Pex11-1p revealed two transmembrane domains with the N- and C-terminus facing the cytoplasm. The C-terminal tail of Pex11-1p ends in a consensus dilysine motif of the type-KXKXX-COOH, which is known to be involved in the ADP-ribosylation factor (ARF)1-coat protein (COP) I coat (ARF)1-dependent membrane recruitment to Golgi membranes. Studies with isolated peroxisomes incubated in the presence of cytosol, adenosine triphosphate and GTPγS, indeed, provided evidence for specific binding of ARF and coatomer to peroxisomes. Expression of Pex11-1p in Chinese hamster ovary (CHO) wild-type cells led to a twofold increase in the number of peroxisomes, but expression in a temperature-sensitive CHO mutant, defective in coatomer, induced elongation and tubulation of peroxisomal structures, rather than numerical proliferation. The obtained results for the first time offer a mechanism explaining Pex11-1p-, as well as ARF- and coatomer-mediated peroxisomal vesiculation. Two models are presented that may explain how these observations fit in with peroxisome biogenesis.     

The peroxisome biogenesis factors pex4p, pex22p, pex1p, and pex6p act in the terminal steps of peroxisomal matrix protein import

Peroxisomes are independent organelles found in virtually all eukaryotic cells. Genetic studies have identified more than 20 PEX genes that are required for peroxisome biogenesis. The role of most PEX gene products, peroxins, remains to be determined, but a variety of studies have established that Pex5p binds the type 1 peroxisomal targeting signal and is the import receptor for most newly synthesized peroxisomal matrix proteins. The steady-state abundance of Pex5p is unaffected in most pex mutants of the yeast Pichia pastoris but is severely reduced in pex4 and pex22 mutants and moderately reduced in pex1 and pex6 mutants. We used these subphenotypes to determine the epistatic relationships among several groups of pex mutants. Our results demonstrate that Pex4p acts after the peroxisome membrane synthesis factor Pex3p, the Pex5p docking factors Pex13p and Pex14p, the matrix protein import factors Pex8p, Pex10p, and Pex12p, and two other peroxins, Pex2p and Pex17p. Pex22p and the interacting AAA ATPases Pex1p and Pex6p were also found to act after Pex10p. Furthermore, Pex1p and Pex6p were found to act upstream of Pex4p and Pex22p. These results suggest that Pex1p, Pex4p, Pex6p, and Pex22p act late in peroxisomal matrix protein import, after matrix protein translocation. This hypothesis is supported by the phenotypes of the corresponding mutant strains. As has been shown previously for P. pastoris pex1, pex6, and pex22 mutant cells, we show here that pex4Delta mutant cells contain peroxisomal membrane protein-containing peroxisomes that import residual amounts of peroxisomal matrix proteins.     

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 Hansenula polymorpha PEX14 gene encodes a novel peroxisomal membrane protein essential for peroxisome biogenesis.

We have cloned the Hansenula polymorpha PEX14 gene by functional complementation of the chemically induced pex14-1 mutant, which lacked normal peroxisomes. The sequence of the PEX14 gene predicts a novel protein product (Pex14p) of 39 kDa which showed no similarity to any known protein and lacked either of the two known peroxisomal targeting signals. Biochemical and electron microscopical analysis indicated that Pex14p is a component of the peroxisomal membrane. The synthesis of Pex14p is induced by peroxisome-inducing growth conditions. In cells of both pex14-1 and a PEX14 disruption mutant, peroxisomal membrane remnants were evident; these contained the H.polymorpha peroxisomal membrane protein Pex3p together with a small amount of the major peroxisomal matrix proteins alcohol oxidase, catalase and dihydroxyacetone synthase, the bulk of which resided in the cytosol. Unexpectedly, overproduction of Pex14p in wild-type H. polymorpha cells resulted in a peroxisome-deficient phenotype typified by the presence of numerous small vesicles which lacked matrix proteins; these were localized in the cytosol. Apparently, the stoichiometry of Pex14p relative to one or more other components of the peroxisome biogenesis machinery appears to be critical for protein import.     

Identification and characterization of the human orthologue of yeast Pex14p

Pex14p is a central component of the peroxisomal protein import machinery, which has been suggested to provide the point of convergence for PTS1- and PTS2-dependent protein import in yeast cells. Here we describe the identification of a human peroxisome-associated protein (HsPex14p) which shows significant similarity to the yeast Pex14p. HsPex14p is a carbonate-resistant peroxisomal membrane protein with its C terminus exposed to the cytosol. The N terminus of the protein is not accessible to exogenously added antibodies or protease and thus might protrude into the peroxisomal lumen. HsPex14p overexpression leads to the decoration of tubular structures and mislocalization of peroxisomal catalase to the cytosol. HsPex14p binds the cytosolic receptor for the peroxisomal targeting signal 1 (PTS1), a result consistent with a function as a membrane receptor in peroxisomal protein import. Homo-oligomerization of HsPex14p or interaction of the protein with the PTS2-receptor or HsPex13p was not observed. This distinguishes the human Pex14p from its counterpart in yeast cells and thus supports recent data suggesting that not all aspects of peroxisomal protein import are conserved between yeasts and humans. The role of HsPex14p in mammalian peroxisome biogenesis makes HsPEX14 a candidate PBD gene for being responsible for an unrecognized complementation group of human peroxisome biogenesis disorders.     

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.     

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.     

Peroxisome biogenesis and peroxisome biogenesis disorders

Peroxisome assembly in mammals requires more than 15 genes. Two isoforms of the peroxisome targeting signal type 1 (PTS1) receptor, Pex5pS and Pex5pL, are identified in mammals. Pex5pS and Pex5pL bind PTS1 proteins. Pex5pL, but not Pex5pS, directly interacts with the PTS2 receptor, Pex7p, carrying its cargo PTS2 protein in the cytosol. Pex5p carrying the cargos, PTS1 and PTS2, docks with the initial site Pex14p in a putative import machinery, subsequently translocating to other components such as Pex13p, Pex2p, Pex10p and Pex12p, whereby the matrix proteins are imported. The peroxins, Pex3p, Pex16p and Pex19p, function in the assembly of peroxisomal membrane vesicles that precedes the import of matrix proteins. Hence, peroxisomes may form de novo and do not have to arise from pre-existing, morphologically recognizable peroxisomes. Impaired peroxisome assembly causes peroxisome biogenesis disorders such as Zellweger syndrome.     

Peroxisome Formation Requires the Endoplasmic Reticulum Channel Protein Sec61

In peroxisome formation, models of near‐autonomous peroxisome biogenesis with membrane protein integration directly from the cytosol into the peroxisomal membrane are in direct conflict with models whereby peroxisomes bud from the endoplasmic reticulum and receive their membrane proteins through a branch of the secretory pathway. We therefore reinvestigated the role of the Sec 61 complex, the protein‐conducting channel of the endoplasmic reticulum (ER) in peroxisome formation. We found that depletion or partial inactivation of Sec 61 in yeast disables peroxisome formation. The ER entry of the early peroxisomal membrane protein Pex 3 engineered with a glycosylation tag is reduced in sec61 mutant cells. Moreover, we were able to reconstitute Pex 3 import into ER membranes in vitro, and we identified a variant of a signal anchor sequence for ER translocation at the Pex 3 N‐terminus. Our findings are consistent with a Sec 61 requirement for peroxisome formation and a fundamental role of the ER in peroxisome biogenesis.     

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.     

Insights into ubiquitin-conjugating enzyme/ co-activator interactions from the structure of the Pex4p:Pex22p complex

Ubiquitin-conjugating enzymes (E2s) coordinate distinct types of ubiquitination via specific E3 ligases, to a large number of protein substrates. While many E2 enzymes need only the presence of an E3 ligase for substrate ubiquitination, a number of E2s require additional, non-canonical binding partners to specify their function. Here, we have determined the crystal structure and function of an E2/co-activator assembly, the Pex4p:Pex22p complex. The peroxisome-associated E2 enzyme Pex4p binds the peroxisomal membrane protein Pex22p through a binding site that does not overlap with any other known interaction interface in E2 enzymes. Pex22p association enhances Pex4p's ability to transfer ubiquitin to a substrate in vitro, and Pex22p binding-deficient forms of Pex4p are unable to ubiquitinate the peroxisomal import receptor Pex5p in vivo. Our data demonstrate that the Pex4p:Pex22p assembly, and not Pex4p alone, functions as the E2 enzyme required for Pex5p ubiquitination, establishing a novel mechanism of E2 enzyme regulation.