PTS1 Receptor Research Articles

Identification of peroxisomal targeting signals in cholesterol biosynthetic enzymes: AA-CoA thiolase, HMG-CoA synthase, MPPD, and FPP synthase

At least three different subcellular compartments, including peroxisomes, are involved in cholesterol synthesis. The peroxisomal targeting signals for phosphomevalonate kinase and isopentenyl diphosphate isomerase have been identified. In the current study we identify the peroxisomal targeting signals required for four other enzymes of the cholesterol biosynthetic pathway: acetoacetyl-CoA (AA-CoA) thiolase, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, mevalonate diphosphate decarboxylase (MPPD), and farnesyl diphosphate (FPP) synthase. Data are presented that demonstrate that mitochondrial AA-CoA thiolase contains both a mitochondrial targeting signal at the amino terminus and a peroxisomal targeting signal (PTS-1) at the carboxy terminus. We also analyze a new variation of PTS-2 sequences required to target HMG-CoA synthase and MPPD to peroxisomes. In addition, we show that FPP synthase import into peroxisomes is dependent on the PTS-2 receptor and identify at the amino terminus of the protein a 20-amino acid region that is required for the peroxisomal localization of the enzyme. These data provide further support for the conclusion that peroxisomes play a critical role in cholesterol biosynthesis.

Identification of a cryptic N-terminal signal in Saccharomyces cerevisiae peroxisomal citrate synthase that functions in both peroxisomal and mitochondrial targeting.

Saccharomyces cerevisiae has three distinct citrate synthases, two located in mitochondria (mature Cit1p and Cit3p) and one in peroxisomes (mature Cit2p). While the precursor of the major mitochondrial enzyme, Cit1p, has a signal for mitochondrial targeting at its N-terminus (MTS), Cit2p has one for peroxisomal targeting (PTS1) at its C-terminus. We have previously shown that the N-terminal segment of Cit2p is removed during import into peroxisomes [Lee, H.S. et al. (1994) Kor. J. Microbiol. 32, 558-564], which implied the presence of an additional N-terminal sorting signal. To analyze the function of the N-terminal region of Cit2p in protein trafficking, we constructed the N-terminal domain-swapped versions of Cit1p and Cit2p. Both fusions, Cit1::Cit2 and Cit2::Cit1, complemented the glutamate auxotrophy caused by the double-disruption of the CIT1 and CIT2 genes. In addition, part of the Cit2::Cit1 fusion protein, as well as Cit1::Cit2, was shown to be transported into both mitochondria and peroxisomes. The subcellular localization of the recombinant fusion proteins containing various N-terminal segments of Cit2p fused to a mutant version of green fluorescent protein (GFP2) was also examined. As a result, we found that the 20-amino acid N-terminal segment of Cit2p contains a cryptic cleavable targeting signal for both peroxisomes and mitochondria. In addition, we show that the peroxisomal import process mediated by the N-terminal segment of Cit2p was not affected by the disruption of either PEX5 (encoding PTS1 receptor) or PEX7 (encoding PTS2 receptor).

Mitochondrial and peroxisomal targeting of 2-methylacyl-CoA racemase in humans

2-Methylacyl-CoA racemase is an auxiliary enzyme required for the peroxisomal β-oxidative breakdown of (2R)-pristanic acid and the (25R)-isomer of C27 bile acid intermediates. The enzyme activity is found not only in peroxisomes but also is present in mitochondria of human liver and fibroblasts. The C terminus of the human racemase, a protein of 382 amino acids with a molecular mass of 43,304 daltons as deduced from its cloned cDNA, consists of KASL. Hitherto this sequence has not been recognized as a peroxisomal targeting signal (PTS1). From the in vitro interaction between recombinant racemase and recombinant human PTS1 receptor (Pex5p), and the peroxisomal localization of green fluorescent protein (GFP) fused to the N terminus of full-length racemase or its last six amino acids in tranfected Chinese hamster ovary (CHO) cells, we concluded that ASL is a new PTS1 variant. To be recognized by Pex5p, however, the preceding lysine residue is critical. As shown in another series of transfection experiments with GFP fused to the C terminus of the full-length racemase or racemase with deletions of the N terminus, mitochondrial targeting information is localized between amino acids 22 and 85. Hence, our data show that a single transcript gives rise to a racemase protein containing two topogenic signals, explaining the dual cellular localization of the activity.—Amery, L., M. Fransen, K. De Nys, G. P. Mannaerts, and P. P. Van Veldhoven. Mitochondrial and peroxisomal targeting of 2-methylacyl-CoA racemase in humans. J. Lipid Res. 2000. 41: 1752–1759.

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.

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.

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.

Overproduction of Pex5p Stimulates Import of Alcohol Oxidase and Dihydroxyacetone Synthase in a Hansenula polymorpha pex14Null 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.

Pex8p, an Intraperoxisomal Peroxin of Saccharomyces cerevisiae Required for Protein Transport into Peroxisomes Binds the PTS1 Receptor Pex5p

We report the characterization of ScPex8p, which is essential for peroxisomal biogenesis in Saccharomyces cerevisiae. Cells lacking Pex8p are characterized by the presence of peroxisomal membrane ghosts and mislocalization of peroxisomal matrix proteins of the PTS1 and PTS2 variety to the cytosol. Pex8p is tightly associated with the lumenal face of the peroxisomal membrane. Consistent with its intraperoxisomal localization, Pex8p contains a peroxisomal targeting signal 1, and it interacts with the PTS1 receptor Pex5p. However, the Pex5p/Pex8p association is also observed upon deletion of the PTS1 of Pex8p, suggesting that Pex8p contains a second binding site for Pex5p. The pex8Delta mutant phenotype and the observed PTS1-independent interaction with the PTS1 receptor suggest that Pex8p is involved in protein import into the peroxisomal matrix. In pex8Delta cells, the PTS1 and PTS2 receptor still associate with membrane bound components of the protein import machinery, supporting the assumption that the Pex8p function in protein translocation follows the docking event.

Peroxisomal matrix protein import. Suppression of protein import defects in Hansenula polymorpha pex mutants by overproduction of the PTS1 receptor Pex5p.

In the past decade, much progress has been made in understanding the mechanisms that govern sorting of proteins to the peroxisomal lumen. This article summarizes the principal features of how peroxisomal matrix enzymes are thought to reach the peroxisome. In addition, it describes recent data that indicate that, in specific pex mutants of the methylotrophic yeast Hansenula polymorpha, defects in matrix protein import can be (partly) rescued by overproduction of the receptor essential for import of these proteins. The implication of these results on the mechanisms of matrix protein import is discussed.

PEX7 Gene Structure, Alternative Transcripts, and Evidence for a Founder Haplotype for the Frequent RCDP Allele, L292ter

We recently reported cloning a cDNA encoding Pex7p, the peroxisomal PTS2 receptor. PEX7 mutations cause the peroxisome biogenesis disorder (PBD) rhizomelic chondrodysplasia punctata (RCDP). In a survey of 44 RCDP probands, we found that one PEX7 allele, L292ter, accounted for 50% of mutant PEX7 genes. Here we report the characterization of the PEX7 structural gene, which spans 102 kb on chromosome 6q21–q22.2 and contains at least 10 exons. In addition to the predominant full-length transcript, we identified eight smaller PEX7 transcripts generated by alternative exon splicing in several tissues. However, none of these splice forms was able to restore PTS2 protein import into peroxisomes when expressed in RCDP fibroblasts nor did they inhibit PTS2 protein import when expressed in normal fibroblasts. To determine whether the high frequency of the L292ter allele is due to a founder effect, we identified five polymorphic markers (four diallelic markers and one CA repeat) spanning the PEX7 gene. We show that all 12 L292ter homozygotes in our patient sample have an identical haplotype at these five sites, consistent with the hypothesis that the L292ter mutation arose once on an ancestral chromosome in the Caucasian population.

PEX12 interacts with PEX5 and PEX10 and acts downstream of receptor docking in peroxisomal matrix protein import.

Peroxisomal matrix protein import requires PEX12, an integral peroxisomal membrane protein with a zinc ring domain at its carboxy terminus. Mutations in human PEX12 result in Zellweger syndrome, a lethal neurological disorder, and implicate the zinc ring domain in PEX12 function. Using two-hybrid studies, blot overlay assays, and coimmunoprecipitation experiments, we observed that the zinc-binding domain of PEX12 binds both PEX5, the PTS1 receptor, and PEX10, another integral peroxisomal membrane protein required for peroxisomal matrix protein import. Furthermore, we identified a patient with a missense mutation in the PEX12 zinc-binding domain, S320F, and observed that this mutation reduces the binding of PEX12 to PEX5 and PEX10. Overexpression of either PEX5 or PEX10 can suppress this PEX12 mutation, providing genetic evidence that these interactions are biologically relevant. PEX5 is a predominantly cytoplasmic protein and previous PEX5-binding proteins have been implicated in docking PEX5 to the peroxisome surface. However, we find that loss of PEX12 or PEX10 does not reduce the association of PEX5 with peroxisomes, demonstrating that these peroxins are not required for receptor docking. These and other results lead us to propose that PEX12 and PEX10 play direct roles in peroxisomal matrix protein import downstream of the receptor docking event.

Cloning and analysis of the PTS-1 receptor in Trypanosoma brucei

Kinetoplastid organisms, such as the protozoan parasite Trypanosoma brucei, compartmentalise several important metabolic pathways in organelles called glycosomes. Glycosomes are related to peroxisomes of yeast and mammalian cells. A subset of glycosomal matrix proteins is routed to the organelles via the peroxisome-targeting signal type 1 (PTS-1). The PEX5 gene homologue has been cloned from T. brucei coding for a protein of the translocation machinery, the PTS-1 receptor. The gene codes for a polypeptide of 654 amino acids with a calculated molecular mass of 70 kDa. Like its homologue in other organisms T. brucei PTS-1 receptor protein (TbPEX5) is a member of the tetratricopeptide repeat (TPR) protein family and contains several copies of the pentapeptide W-X-X-X-F/Y. Northern and Western blot analysis showed that the protein is expressed at different stages of the life cycle of the parasite. The protein has been overproduced in Escherichia coli and purified using immobilized metal affinity chromatography. The purified protein specifically interacts in vitro with glycosomal phosphoglycerate kinase-C (PGK-C) of T. brucei, a PTS-1 containing protein. The equilibrium dissociation constant ( K d) of PGK-C for purified TbPEX5 is 40 nM. Using biochemical and cytochemical techniques a predominantly cytosolic localization was found for TbPEX5. This is consistent with the idea of receptor cycling between the glycosomes and the cytosol.

Functional Heterogeneity of C-Terminal Peroxisome Targeting Signal 1 in PEX5-Defective Patients

To investigate mechanisms related to functions of the peroxisome targeting signal (PTS) 1 receptor, Pex5p, we analyzed peroxisome matrix protein import in fibroblasts from three patients with peroxisome biogenesis disorders, all with different mutations in the PEX5 gene. The patients 2-01 (Zellweger syndrome) and 2-05 (neonatal adrenoleukodystrophy) have the reported mutations, R390X and N489K, and patient 2-03 (infantile Refsum disease) has a newly identified mutation, S563W. Fibroblasts from 2-03 (S563W) were detected in both PTS1 and PTS2 imports despite the PEX5 defect, findings in contrast with fibroblasts from 2-05 (N489K) severely defective in PTS1 import and those from 2-01 (R390X) severely defective in both PTS1 and PTS2. The PTS1 receptor in 2-03 is functional for only the C-terminal -SKL sequence (acyl-CoA oxidase) and had little or no function for C-terminal -AKL (D-bifunctional protein and sterol carrier protein 2) and -KANL (catalase) sequences, respectively. After transfection of these mutated PEX5 cDNA into the PEX5-defective CHO mutant, transformants of ZP102 revealed that each mutation was responsible for each dysfunction of the PTS1 import. It seems apparent that -AKL and -KANL are poorer variants of PTS1 and are likely to be more susceptible to effects of mutation of its receptor, Pex5p.

Recombinant Human Peroxisomal Targeting Signal Receptor PEX5: STRUCTURAL BASIS FOR INTERACTION OF PEX5 WITH PEX14

Import of matrix proteins into peroxisomes requires two targeting signal-specific import receptors, Pex5p and Pex7p, and their binding partners at the peroxisomal membrane, Pex13p and Pex14p. Several constructs of human PEX5 have been overexpressed and purified by affinity chromatography in order to determine functionally important interactions and provide initial structural information. Sizing chromatography and electron microscopy suggest that the two isoforms of the human PTS1 receptor, PEX5L and PEX5S, form homotetramers. Surface plasmon resonance analysis indicates that PEX5 binds to the N-terminal fragment of PEX14-(1-78) with a very high affinity in the low nanomolar range. Stable complexes between recombinant PEX14-(1-78) and both the full-length and truncated versions of PEX5 were formed in vitro. Analysis of these complexes revealed that PEX5 possesses multiple binding sites for PEX14, which appear to be distributed throughout its N-terminal half. Coincidentally, this part of the molecule is also responsible for oligomerization, whereas the C-terminal half with its seven tetratricopeptide repeats has been reported to bind PTS1-proteins. A pentapeptide motif that is reiterated seven times in PEX5 is proposed as a determinant for the interaction with PEX14.

Genomic Structure ofPEX13,a Candidate Peroxisome Biogenesis Disorder Gene

The peroxisome biogenesis disorders (PBDs) are a set of lethal genetic diseases characterized by peroxisomal metabolic deficiencies, multisystem abnormalities, mental retardation, and premature death. These disorders are genetically heterogeneous and are caused by mutations in genes, termedPEXgenes, required for import of proteins into the peroxisomal matrix. We have previously reported the identification of humanPEX13,the gene encoding the docking factor for the PTS1 receptor, or PEX5 protein. As such, mutations inPEX13would be expected to abrogate peroxisomal protein import and result in PBD phenotypes. We report here the structure of the humanPEX13gene.PEX13spans approximately 11 kb on chromosome 2 and contains four exons, one more than previously thought. The corrected PEX13 cDNA is predicted to encode a protein product with a molecular mass of 44,312 Da. We examined the ability ofPEX13expression to rescue the peroxisomal protein import defects of fibroblast cells representing all known PBD complementation groups. No complementation was observed, suggesting that this gene is not mutated in any set of existing patients. However, given that complementation group assignments have been determined for only a subset of PBD patients, it is possible thatPEX13-deficient patients may exist at a low frequency within our existing PBD patient population or within ethnic groups underrepresented in our patient pool.

The plant PTS1 receptor: similarities and differences to its human and yeast counterparts.

Two targeting signals, PTS1 and PTS2, mediate import of proteins into the peroxisomal matrix. We have cloned and sequenced the watermelon (Citrullus vulgaris) cDNA homologue to the PTS1 receptor gene (PEX5). Its gene product, CvPex5p, belongs to the family of tetratricopeptide repeat (TPR) containing proteins like the human and yeast counterparts, and exhibits 11 repeats of the sequence W-X2-(E/S)-(Y/F/Q) in its N-terminal half. According to fractionation studies the plant Pex5p is located mainly in the cytosolic fraction and therefore could function as a cycling receptor between the cytosol and glyoxysomes, as has been proposed for the Pex5p of human and some yeast peroxisomes. Transformation of the Hansenula polymorpha peroxisome deficient pex5 mutant with watermelon PEX5 resulted in restoration of peroxisome formation and the synthesis of additional membranes surrounding the peroxisomes. These structures are labeled in immunogold experiments using antibodies against the Hansenula polymorpha integral membrane protein Pex3p, confirming their peroxisomal nature. The plant Pex5p was localized by immunogold labelling mainly in the cytosol of the yeast, but also inside the newly formed peroxisomes. However, import of the PTS1 protein alcohol oxidase is only partially restored by CvPex5p.

Identification and analysis of the plant peroxisomal targeting signal 1 receptor NtPEX5.

Protein translocation into peroxisomes takes place via recognition of a peroxisomal targeting signal present at either the extreme C termini (PTS1) or N termini (PTS2) of matrix proteins. In mammals and yeast, the peroxisomal targeting signal receptor, Pex5p, recognizes the PTS1 consisting of -SKL or variants thereof. Although many plant peroxisomal matrix proteins are transported through the PTS1 pathway, little is known about the PTS1 receptor or any other peroxisome assembly protein from plants. We cloned tobacco (Nicotiana tabacum) cDNAs encoding Pex5p (NtPEX5) based on the protein's interaction with a PTS1-containing protein in the yeast two-hybrid system. Nucleotide sequence analysis revealed that the tobacco Pex5p contains seven tetratricopeptide repeats and that NtPEX5 shares greater sequence similarity with its homolog from humans than from yeast. Expression of NtPEX5 fusion proteins, consisting of the N-terminal part of yeast Pex5p and the C-terminal region of NtPEX5, in a Saccharomyces cerevisiae pex5 mutant restored protein translocation into peroxisomes. These experiments confirmed the identity of the tobacco protein as a PTS1 receptor and indicated that components of the peroxisomal translocation apparatus are conserved functionally. Two-hybrid assays showed that NtPEX5 interacts with a wide range of PTS1 variants that also interact with the human Pex5p. Interestingly, the C-terminal residues of some of these peptides deviated from the established plant PTS1 consensus sequence. We conclude that there are significant sequence and functional similarities between the plant and human Pex5ps.

An isoform of pex5p, the human PTS1 receptor, is required for the import of PTS2 proteins into peroxisomes.

Mutations in the peroxisome targeting signal (PTS) 1 receptor gene, PEX5 , are responsible for complementation group (CG) 2 of the peroxisome biogenesis disorders (PBD). Of the two reported patients in this CG, cells from PBD018 (homozygous for the missense mutation N489K) are defective in the import of PTS1 proteins into peroxisomes, as expected. However, cells from PBD005 (homozygous for the nonsense mutation R390ter) are defective in the import of both PTS1 and PTS2 proteins, suggesting that the PTS1 receptor also mediates PTS2-targeted protein import. To investigate this possibility, we characterized PEX5 expression and found that it undergoes alternative splicing, producing two transcripts, one containing (PEX5L) and one lacking (PEX5S) a 111 bp internal exon. Fibroblasts from PBD005 have greatly reduced levels of PEX5 transcript and protein as compared with PBD018. Transfection of PBD005 cells with PEX5S cDNA restores PTS1 but not PTS2 import; transfection with PXR5L cDNA restores both PTS1 and PTS2 protein import. Furthermore, transfection of PBD005 cells with PEX5L cDNAs containing the patient mutations (which are located downstream of the additional exon) restores PTS2 but not PTS1 import. Taken together, these data provide an explanation for the different protein import defects in CG2 patients and show that the long isoform of the Pex5 protein is required for peroxisomal import of PTS2 proteins.

Identification of a human PTS1 receptor docking protein directly required for peroxisomal protein import.

The discovery of many fatal human disorders resulting from impaired peroxisomal protein import makes the functional characterization of human peroxins critical. As part of our attempt to identify novel human genes and gene products involved in the import of peroxisomal proteins, we raised antisera against peroxisomal membrane proteins. One such antiserum inhibited peroxisomal protein import in semipermeabilized mammalian cells. This "import inhibiting" antiserum, ab-MF3, specifically recognized a 57-kDa protein. Immunoblot analysis of rat liver subcellular fractions demonstrated that this protein was present exclusively in peroxisomal membranes. Functional analysis revealed that this 57-kDa molecule bound the PTS1 receptor, Pex5p, in ligand blots, suggesting it is a docking site on the peroxisomal membrane. Previous studies have identified two yeast proteins, Pex14p and Pex13p, as Pex5p-binding proteins. To facilitate the biochemical analysis of peroxisomal membrane docking proteins, we cloned and expressed the previously unidentified human Pex14p, as well as a human Pex13p that is 39 aa longer than previously reported. Recombinant Pex14p was specifically recognized by the "import inhibiting" ab-MF3 and bound Pex5p and the Src homology 3 (SH3) domain of Pex13p in ligand blots. These studies demonstrate that the ab-MF3-immunoreactive, 57-kDa peroxisomal membrane protein is Pex14p. Furthermore, this peroxin interacts with Pex5p and Pex13p(SH3) and is directly required for peroxisomal protein import.

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.