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.     

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.     

Ubiquitination of the peroxisomal targeting signal type 1 receptor, Pex5p, suggests the presence of a quality control mechanism during peroxisomal matrix protein import

PEX genes encode proteins (peroxins) that are required for the biogenesis of peroxisomes. One of these peroxins, Pex5p, is the receptor for matrix proteins with a type 1 peroxisomal targeting signal (PTS1), which shuttles newly synthesized proteins from the cytosol into the peroxisome matrix. We observed that in various Saccharomyces cerevisiae pex mutants disturbed in the early stages of PTS1 import, the steady-state levels of Pex5p are enhanced relative to wild type controls. Furthermore, we identified ubiquitinated forms of Pex5p in deletion mutants of those PEX genes that have been implicated in recycling of Pex5p from the peroxisomal membrane into the cytosol. Pex5p ubiquitination required the presence of the ubiquitin-conjugating enzyme Ubc4p and the peroxins that are required during early stages of PTS1 protein import. Finally, we provide evidence that the proteasome is involved in the turnover of Pex5p in wild type yeast cells, a process that requires Ubc4p and occurs at the peroxisomal membrane. Our data suggest that during receptor recycling a portion of Pex5p becomes ubiquitinated and degraded by the proteasome. We propose that this process represents a conserved quality control mechanism in peroxisome biogenesis.     

Pex17p of Saccharomyces cerevisiae Is a Novel Peroxin and Component of the Peroxisomal Protein Translocation Machinery

The Saccharomyces cerevisiae pex17-1 mutant was isolated from a screen to identify mutants defective in peroxisome biogenesis. pex17-1 and pex17 null mutants fail to import matrix proteins into peroxisomes via both PTS1- and PTS2-dependent pathways. The PEX17 gene (formerly PAS9; Albertini, M., P. Rehling, R. Erdmann, W. Girzalsky, J.A.K.W. Kiel, M. Veenhuis, and W.-H Kunau. 1997. Cell. 89:83–92) encodes a polypeptide of 199 amino acids with one predicted membrane spanning region and two putative coiled-coil structures. However, localization studies demonstrate that Pex17p is a peripheral membrane protein located at the surface of peroxisomes. Particulate structures containing the peroxisomal integral membrane proteins Pex3p and Pex11p are evident in pex17 mutant cells, indicating the existence of peroxisomal remnants (“ghosts”). This finding suggests that pex17 null mutant cells are not impaired in peroxisomal membrane biogenesis. Two-hybrid studies showed that Pex17p directly binds to Pex14p, the recently proposed point of convergence for the two peroxisomal targeting signal (PTS)-dependent import pathways, and indirectly to Pex5p, the PTS1 receptor. The latter interaction requires Pex14p, indicating the potential of these three peroxins to form a trimeric complex. This conclusion is supported by immunoprecipitation experiments showing that Pex14p and Pex17p coprecipitate with both PTS receptors in the absence of Pex13p. From these and other studies we conclude that Pex17p, in addition to Pex13p and Pex14p, is the third identified component of the peroxisomal translocation machinery.     

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.     

The ubiquitin-conjugating enzyme Pex4p of Hansenula polymorpha is required for efficient functioning of the PTS1 import machinery.

We have cloned the Hansenula polymorpha PEX4 gene by functional complementation of a peroxisome-deficient mutant. The PEX4 translation product, Pex4p, is a member of the ubiquitin-conjugating enzyme family. In H.polymorpha, Pex4p is a constitutive, low abundance protein. Both the original mutant and the pex4 deletion strain (Deltapex4) showed a specific defect in import of peroxisomal matrix proteins containing a C-terminal targeting signal (PTS1) and of malate synthase, whose targeting signal is not yet known. Import of the PTS2 protein amine oxidase and the insertion of the peroxisomal membrane proteins Pex3p and Pex14p was not disturbed in Deltapex4 cells. The PTS1 protein import defect in Deltapex4 cells could be suppressed by overproduction of the PTS1 receptor, Pex5p, in a dose-response related manner. In such cells, Pex5p is localized in the cytosol and in peroxisomes. The peroxisome-bound Pex5p specifically accumulated at the inner surface of the peroxisomal membrane and thus differed from Pex5p in wild-type peroxisomes, which is localized throughout the matrix. We hypothesize that in H. polymorpha Pex4p plays an essential role for normal functioning of Pex5p, possibly in mediating recycling of Pex5p from the peroxisome to the cytosol.     

Disruption of the interaction of the longer isoform of Pex5p, Pex5pL, with Pex7p abolishes peroxisome targeting signal type 2 protein import in mammals. Study with a novel Pex5-impaired Chinese hamster ovary cell mutant

We isolated peroxisome biogenesis-defective Chinese hamster ovary cell mutants from TKaG2 cells, wild-type CHO-K1 cells transformed with two cDNAs encoding rat Pex2p and peroxisome targeting signal (PTS) type 2-tagged green fluorescent protein, by the 9-(1'-pyrene)nonanol/UV selection method. Ten mutant clones showed cytosolic PTS2-green fluorescent protein, indicative of a defect in PTS2 import, and were classified in five complementation groups, i.e. pex1, pex2, pex5, pex14, and group A. One PEX5-deficient mutant, ZPG231, showed a novel phenotype: PTS2 proteins in the cytosol, but PTS1 proteins and catalase in peroxisomes. In ZPG231, two isoforms of the PTS1 receptor Pex5p, a shorter Pex5pS and a longer Pex5pL, were expressed as in wild-type cells, but possessed the missense point mutation S214F in both Pex5p isoforms, termed Pex5pS-S214F and Pex5pL-S214F, respectively. The S214F mutation was located only one amino acid upstream of the Pex5pL-specific 37-amino acid insertion site. Pex5pS-S214F and Pex5pL-S214F interacted with peroxisomal proteins, including PTS1 protein, catalase, and Pex14p, as efficiently as normal Pex5p. In contrast, the S214F mutation severely affected the binding of Pex5pL to the PTS2 receptor Pex7p. Expression of Pex5pL-S214F in pex5 cell mutants defective in PTS1 and PTS2 transport restored peroxisomal import of PTS1, but not PTS2. Together, the results indicate that ZPG231 is the first cell mutant providing evidence that disruption of the Pex5pL-Pex7p interaction completely abolishes PTS2 import in mammals.     

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.     

Overexpression of Pex15p, a phosphorylated peroxisomal integral membrane protein required for peroxisome assembly in S.cerevisiae, causes proliferation of the endoplasmic reticulum membrane.

We have cloned PEX15 which is required for peroxisome biogenesis in Saccharomyces cerevisiae. pex15Delta cells are characterized by the cytosolic accumulation of peroxisomal matrix proteins containing a PTS1 or PTS2 import signal, whereas peroxisomal membrane proteins are present in peroxisomal remnants. PEX15 encodes a phosphorylated, integral peroxisomal membrane protein (Pex15p). Using multiple in vivo methods to determine the topology, Pex15p was found to be a tail-anchored type II (Ncyt-Clumen) peroxisomal membrane protein with a single transmembrane domain near its carboxy-terminus. Overexpression of Pex15p resulted in impaired peroxisome assembly, and caused profound proliferation of the endoplasmic reticulum (ER) membrane. The lumenal carboxy-terminal tail of Pex15p protrudes into the lumen of these ER membranes, as demonstrated by its O-glycosylation. Accumulation in the ER was also observed at an endogenous expression level when Pex15p was fused to the N-terminus of mature invertase. This resulted in core N-glycosylation of the hybrid protein. The lumenal C-terminal tail of Pex15p is essential for targeting to the peroxisomal membrane. Furthermore, the peroxisomal membrane targeting signal of Pex15p overlaps with an ER targeting signal on this protein. These results indicate that Pex15p may be targeted to peroxisomes via the ER, or to both organelles.     

Penicillium chrysogenum Pex5p mediates differential sorting of PTS1 proteins to microbodies of the methylotrophic yeast Hansenula polymorpha

We have isolated the Penicillium chrysogenum pex5 gene encoding the receptor for microbody matrix proteins containing a type 1 peroxisomal targeting signal (PTS1). Pc-pex5 contains 2 introns and encodes a protein of approximately 75 kDa. P. chrysogenum pex5 disruptants appear to be highly unstable, show poor growth, and are unable to sporulate asexually. Furthermore, pex5 cells mislocalize a fluorescent PTS1 reporter protein to the cytosol. Pc-pex5 was expressed in a PEX5 null mutant of the yeast Hansenula polymorpha. Detailed analysis demonstrated that the PTS1 proteins dihydroxyacetone synthase and catalase were almost fully imported into microbodies. Surprisingly, alcohol oxidase, which also depends on Pex5p for import into microbodies, remained mainly in the cytosol. Thus, P. chrysogenum Pex5p has a different specificity of cargo recognition than its H. polymorpha counterpart. This was also suggested by the observation that Pc-Pex5p sorted a reporter protein fused to various functional PTS1 signals with different efficiencies.     

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 Saccharomyces cerevisiae peroxisomal import receptor Pex5p is monoubiquitinated in wild type cells

Pex5p is a mobile receptor for peroxisomal targeting signal type I-containing proteins that cycles between the cytoplasm and the peroxisome. Here we show that Pex5p is a stable protein that is monoubiquitinated in wild type cells. By making use of mutants defective in vacuolar or proteasomal degradation we demonstrate that monoubiquitinated Pex5p is not a breakdown intermediate of either system. Monoubiquitinated Pex5p is localized to peroxisomes, and ubiquitination requires the presence of functional docking and RING finger complexes, which suggests that it is a late event in peroxisomal matrix protein import. In pex1, pex4, pex6, pex15, and pex22 mutants, all of which are blocked in the terminal steps of peroxisomal matrix protein import, polyubiquitinated forms of Pex5p accumulate, ubiquitination being dependent on the ubiquitin-conjugating enzyme Ubc4p. However, Ubc4p is not required for Pex5p ubiquitination in wild type cells, and cells lacking Ubc4p are not affected in peroxisome biogenesis. These results indicate that Pex5p monoubiquitination in wild type cells serves to regulate rather than to degrade Pex5p, which is supported by the observed stability of Pex5p. We propose that Pex5p monoubiquitination in wild type cells is required for the recycling of Pex5p from the peroxisome, whereas Ubc4p-mediated polyubiquitination of Pex5p in mutants blocked in the terminal steps of peroxisomal matrix protein import may function as a disposal mechanism for Pex5p when it gets stuck in the import pathway.     

Overproduction of Pex5p stimulates import of alcohol oxidase and dihydroxyacetone synthase in a Hansenula polymorpha Pex14 null mutant

Hansenula polymorpha Deltapex14 cells are affected in peroxisomal matrix protein import and lack normal peroxisomes. Instead, they contain peroxisomal membrane remnants, which harbor a very small amount of the major peroxisomal matrix enzymes alcohol oxidase (AO) and dihydroxyacetone synthase (DHAS). The bulk of these proteins is, however, mislocated in the cytosol. Here, we show that in Deltapex14 cells overproduction of the PTS1 receptor, Pex5p, leads to enhanced import of the PTS1 proteins AO and DHAS but not of the PTS2 protein amine oxidase. The import of the PTS1 protein catalase (CAT) was not stimulated by Pex5p overproduction. The difference in import behavior of AO and CAT was not related to their PTS1, since green fluorescent protein fused to the PTS1 of either AO or CAT were both not imported in Deltapex14 cells overproducing Pex5p. When produced in a wild type control strain, both proteins were normally imported into peroxisomes. In Deltapex14 cells overproducing Pex5p, Pex5p had a dual location and was localized in the cytosol and bound to the outer surface of the peroxisomal membrane. Our results indicate that binding of Pex5p to the peroxisomal membrane and import of certain PTS1 proteins can proceed in the absence of Pex14p.     

Ubp15p, a ubiquitin hydrolase associated with the peroxisomal export machinery

Peroxisomal matrix protein import is facilitated by cycling receptors shuttling between the cytosol and the peroxisomal membrane. One crucial step in this cycle is the ATP-dependent release of the receptors from the peroxisomal membrane. This step is facilitated by the peroxisomal AAA (ATPases associated with various cellular activities) proteins Pex1p and Pex6p with ubiquitination of the receptor being the main signal for its export. Here we report that the AAA complex contains dislocase as well as deubiquitinating activity. Ubp15p, a ubiquitin hydrolase, was identified as a novel constituent of the complex. Ubp15p partially localizes to peroxisomes and is capable of cleaving off ubiquitin moieties from the type I peroxisomal targeting sequence (PTS1) receptor Pex5p. Furthermore, Ubp15p-deficient cells are characterized by a stress-related PTS1 import defect. The results merge into a picture in which removal of ubiquitin from the PTS1 receptor Pex5p is a specific event and might represent a vital step in receptor recycling.     

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.     

Yeast Pex14p possesses two functionally distinct Pex5p and one Pex7p binding sites

Current evidence favors a cycling receptor model for the import of peroxisomal matrix proteins. The yeast Pex14 protein together with Pex13p and Pex17p form the docking subcomplex at the peroxisomal membrane and interact in this cycle with both soluble import receptors Pex5p and Pex7p. In a first step of a structure-function analysis of Saccharomyces cerevisiae Pex14p, we mapped its binding sites with both receptors. Using the yeast two-hybrid system and pull-down assays, we showed that Pex5p directly interacts with two separate regions of ScPex14p, amino acid residues 1-58 and 235-308. The latter binding site at the C terminus of ScPex14p overlaps with a binding site of Pex7p at amino acid residues 235-325. The functional assessment of these two binding sites of ScPex14p with the peroxisomal targeting signal receptors indicates that they have distinct roles. Deletion of the N-terminal 58 amino acids caused a partial defect of matrix protein import in pex14delta cells expressing the Pex14-(59-341)-p fragment; however, it did not lead to a pex phenotype. In contrast, truncation of the C-terminal 106 amino acids of ScPex14p completely blocked this process. On the basis of these and other published data, we propose that the C terminus of Pex14p contains the actual docking site and discuss the possibility that the N terminus could be involved in a Pex5p-Pex14p association inside the peroxisomal membrane.     

Pex13p is an SH3 protein of the peroxisome membrane and a docking factor for the predominantly cytoplasmic PTs1 receptor

Import of newly synthesized PTS1 proteins into the peroxisome requires the PTS1 receptor (Pex5p), a predominantly cytoplasmic protein that cycles between the cytoplasm and peroxisome. We have identified Pex13p, a novel integral peroxisomal membrane from both yeast and humans that binds the PTS1 receptor via a cytoplasmically oriented SH3 domain. Although only a small amount of Pex5p is bound to peroxisomes at steady state (< 5%), loss of Pex13p further reduces the amount of peroxisome- associated Pex5p by approximately 40-fold. Furthermore, loss of Pex13p eliminates import of peroxisomal matrix proteins that contain either the type-1 or type-2 peroxisomal targeting signal but does not affect targeting and insertion of integral peroxisomal membrane proteins. We conclude that Pex13p functions as a docking factor for the predominantly cytoplasmic PTS1 receptor.     

The tetratricopeptide repeat domains of human, tobacco, and nematode PEX5 proteins are functionally interchangeable with the analogous native domain for peroxisomal import of PTS1-terminated proteins in yeast

In the yeast Saccharomyces cerevisiae, beta-oxidation of fatty acids is compartmentalised in peroxisomes. Most yeast peroxisomal matrix proteins contain a type 1C-terminal peroxisomal targeting signal (PTS1) consisting of the tripeptide SKL or a conservative variant thereof. PTS1-terminated proteins are imported by Pex5p, which interacts with the targeting signal via a tetratricopeptide repeat (TPR) domain. Yeast cells devoid of Pex5p are unable to import PTS1-containing proteins and cannot degrade fatty acids. Here, the PEX5-TPR domains from human, tobacco, and nematode were inserted into a TPR-less yeast Pex5p construct to generate Pex5p chimaeras. These hybrid proteins were examined for functional complementation of the pex5delta mutant phenotype. Expression of the Pex5p chimaeras in pex5delta mutant cells restored peroxisomal import of PTS1-terminated proteins. Chimaera expression also re-established degradation of oleic acid, allowing growth on this fatty acid as a sole carbon source. We conclude that, in the context of Pex5p chimaeras, the human, tobacco, and nematode Pex5p-TPR domains are functionally interchangeable with the native domain for the peroxisomal import of yeast proteins terminating with canonical PTS1s. Non-conserved yeast PTS1s, such as HRL and HKL, did not interact with the tobacco PEX5-TPR domain in the two-hybrid system. HRL occurs at the C-terminus of the peroxisomal protein Eci1p, which is required for growth on unsaturated fatty acids. Although mutant pex5delta cells expressing a yeast/tobacco Pex5p chimaera failed to import a GFP-Eci1p reporter protein, they were able to grow on oleic acid. We reason that this is due to a cryptic PTS in native Eci1p that can function in a redundant system with the C-terminal HRL.     

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.     

Interactions of Pex7p and Pex18p/Pex21p with the peroxisomal docking machinery: implications for the first steps in PTS2 protein import

Peroxisomal PTS2-dependent matrix protein import starts with the recognition of the PTS2 targeting signal by the import receptor Pex7p. Subsequently, the formed Pex7p/cargo complex is transported from the cytosol to the peroxisomal docking complex, consisting of Pex13p and Pex14p. In Saccharomyces cerevisiae, the latter event is thought to require the redundant Pex18p and Pex21p. Here we mapped the Pex7p interaction domain of Pex13p to its N-terminal 100 amino acids. Pex18p and Pex21p also interacted with this region, albeit only in the presence of Pex7p. Expression of an N-terminally deleted version of Pex13p in a pex13delta mutant failed to restore growth on fatty acids due to a specific defect in the import of PTS2-containing proteins. We further show by yeast two-hybrid analysis, coimmunoprecipitation, and in vitro binding assays that Pex7p can bind Pex13p and Pex14p in the absence of Pex18p/Pex21p. The PTS2 protein thiolase was shown to interact with Pex14p but not with Pex13p in a Pex7p- and Pex18p/Pex21p-dependent manner, suggesting that only Pex14p binds cargo-loaded PTS2 receptor. We also found that the cytosolic Pex7p/thiolase-containing complex includes Pex18p. This complex accumulated in docking mutants but was absent in cells lacking Pex18p/Pex21p, indicating that Pex18p/Pex21p are required already before the docking event.