The peroxin Pex17p of the yeast Yarrowia lipolytica is associated peripherally with the peroxisomal membrane and is required for the import of a subset of matrix proteins.

PEX genes encode peroxins, which are required for the biogenesis of peroxisomes. The Yarrowia lipolytica PEX17 gene encodes the peroxin Pex17p, which is 671 amino acids in length and has a predicted molecular mass of 75,588 Da. Pex17p is peripherally associated with the peroxisomal membrane. The carboxyl-terminal tripeptide, Gly-Thr-Leu, of Pex17p is not necessary for its targeting to peroxisomes. Synthesis of Pex17p is low in cells grown in glucose-containing medium and increases after the cells are shifted to oleic acid-containing medium. Cells of the pex17-1 mutant, the original mutant strain, and the pex17-KA mutant, a strain in which most of the PEX17 gene is deleted, fail to form normal peroxisomes but instead contain numerous large, multimembraned structures. The import of peroxisomal matrix proteins in these mutants is selectively impaired. This selective import is not a function of the nature of the peroxisomal targeting signal. We suggest a regulatory role for Pex17p in the import of a subset of matrix proteins into peroxisomes.     

A role for the peroxin Pex8p in Pex20p-dependent thiolase import into peroxisomes of the yeast Yarrowia lipolytica

Peroxins are proteins required for peroxisome assembly. The cytosolic peroxin Pex20p binds directly to the beta-oxidation enzyme thiolase and is necessary for its dimerization and peroxisomal targeting. The intraperoxisomal peroxin Pex8p has a role in the import of peroxisomal matrix proteins, including thiolase. We report the results of yeast two-hybrid analyses with various peroxins of the yeast Yarrowia lipolytica and characterize more fully the interaction between Pex8p and Pex20p. Coimmunoprecipitation showed that Pex8p and Pex20p form a complex, while in vitro binding studies demonstrated that the interaction between Pex8p and Pex20p is specific, direct, and autonomous. Pex8p fractionates with peroxisomes in cells of a PEX20 disruption strain, indicating that Pex20p is not necessary for the targeting of Pex8p to peroxisomes. In cells of a PEX8 disruption strain, thiolase is mostly cytosolic, while Pex20p and a small amount of thiolase associate with peroxisomes, suggesting the involvement of Pex8p in the import of thiolase after docking of the Pex20p-thiolase complex to the membrane. In the absence of Pex8p, peroxisomal thiolase and Pex20p are protected from the action of externally added protease. This finding, together with the fact that Pex8p is intraperoxisomal, suggests that Pex20p may accompany thiolase into peroxisomes during import.     

Yarrowia lipolytica Cells Mutant for the Peroxisomal Peroxin Pex19p Contain Structures Resembling Wild-Type Peroxisomes

PEX genes encode peroxins, which are proteins required for peroxisome assembly. The PEX19 gene of the yeast Yarrowia lipolytica was isolated by functional complementation of the oleic acid-nonutilizing strain pex19-1 and encodes Pex19p, a protein of 324 amino acids (34,822 Da). Subcellular fractionation and immunofluorescence microscopy showed Pex19p to be localized primarily to peroxisomes. Pex19p is detected in cells grown in glucose-containing medium, and its levels are not increased by incubation of cells in oleic acid-containing medium, the metabolism of which requires intact peroxisomes. pex19 cells preferentially mislocalize peroxisomal matrix proteins and the peripheral intraperoxisomal membrane peroxin Pex16p to the cytosol, although small amounts of these proteins could be reproducibly localized to a subcellular fraction enriched for peroxisomes. In contrast, the peroxisomal integral membrane protein Pex2p exhibits greatly reduced levels in pex19 cells compared with its levels in wild-type cells. Importantly, pex19 cells were shown by electron microscopy to contain structures that resemble wild-type peroxisomes in regards to size, shape, number, and electron density. Subcellular fractionation and isopycnic density gradient centrifugation confirmed the presence of vesicular structures in pex19 mutant strains that were similar in density to wild-type peroxisomes and that contained profiles of peroxisomal matrix and membrane proteins that are similar to, yet distinct from, those of wild-type peroxisomes. Because peroxisomal structures form in pex19 cells, Pex19p apparently does not function as a peroxisomal membrane protein receptor in Y. lipolytica. Our results are consistent with a role for Y. lipolytica Pex19p in stabilizing the peroxisomal membrane.     

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

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

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.     

Molecular anatomy of the peroxin Pex12p: ring finger domain is essential for Pex12p function and interacts with the peroxisome-targeting signal type 1-receptor Pex5p and a ring peroxin, Pex10p

The three peroxin genes, PEX12, PEX2, and PEX10, encode peroxisomal integral membrane proteins with RING finger at the C-terminal part and are responsible for human peroxisome biogenesis disorders. Mutation analysis in PEX12 of Chinese hamster ovary cell mutants revealed a homozygous nonsense mutation at residue Trp263Ter in ZP104 cells and a pair of heterozygous nonsense mutations, Trp170Ter and Trp114Ter, in ZP109. This result and domain mapping of Pex12p showed that RING finger is essential for peroxisome-restoring activity of Pex12p but not necessary for targeting to peroxisomes. The N-terminal region of Pex12p, including amino acid residues at positions 17-76, was required for localization to peroxisomes, while the sequence 17-76 was not sufficient for peroxisomal targeting. Peroxins interacting with RING finger of Pex2p, Pex10p, and Pex12p were investigated by yeast two-hybrid as well as in vitro binding assays. The RING finger of Pex12p bound to Pex10p and the PTS1-receptor Pex5p. Pex10p also interacted with Pex2p and Pex5p in vitro. Moreover, Pex12p was co-immunoprecipitated with Pex10p from CHO-K1 cells, where Pex5p was not associated with the Pex12p-Pex10p complex. This observation suggested that Pex5p does not bind to, or only transiently interacts with, Pex10p and Pex12p when Pex10p and Pex12p are in the oligomeric complex in peroxisome membranes. Hence, the RING finger peroxins are most likely to be involved in Pex5p-mediated matrix protein import into peroxisomes.     

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.     

YHR150w and YDR479c encode peroxisomal integral membrane proteins involved in the regulation of peroxisome number,size, and distribution in Saccharomyces cerevisiae

The peroxin Pex24p of the yeast Yarrowia lipolytica exhibits high sequence similarity to two hypothetical proteins, Yhr150p and Ydr479p, encoded by the Saccharomyces cerevisiae genome. Like YlPex24p, both Yhr150p and Ydr479p have been shown to be integral to the peroxisomal membrane, but unlike YlPex24p, their levels of synthesis are not increased upon a shift of cells from glucose- to oleic acid-containing medium. Peroxisomes of cells deleted for either or both of the YHR150w and YDR479c genes are increased in number, exhibit extensive clustering, are smaller in area than peroxisomes of wild-type cells, and often exhibit membrane thickening between adjacent peroxisomes in a cluster. Peroxisomes isolated from cells deleted for both genes have a decreased buoyant density compared with peroxisomes isolated from wild-type cells and still exhibit clustering and peroxisomal membrane thickening. Overexpression of the genes PEX25 or VPS1, but not the gene PEX11, restored the wild-type phenotype to cells deleted for one or both of the YHR150w and YDR479c genes. Together, our data suggest a role for Yhr150p and Ydr479p, together with Pex25p and Vps1p, in regulating peroxisome number, size, and distribution in S. cerevisiae. Because of their role in peroxisome dynamics, YHR150w and YDR479c have been designated as PEX28 and PEX29, respectively, and their encoded peroxins as Pex28p and Pex29p.     

Pex14/17, a filamentous fungus‐specific peroxin,is required for the import of peroxisomal matrix proteins and full virulence of Magnaporthe oryzae

Peroxisomes are ubiquitous organelles in eukaryotic cells that fulfil a variety of biochemical functions. The biogenesis of peroxisomes requires a variety of proteins, named peroxins, which are encoded by PEX genes. Pex14/17 is a putative recently identified peroxin, specifically present in filamentous fungal species. Its function in peroxisomal biogenesis is still obscure and its roles in fungal pathogenicity have not yet been documented. Here, we demonstrate the contributions of Pex14/17 in the rice blast fungus Magnaporthe oryzae (Mopex14/17) to peroxisomal biogenesis and fungal pathogenicity by targeting gene replacement strategies. Mopex14/17 has properties of both Pex14 and Pex17 with regard to its protein sequence. Mopex14/17 is distributed at the peroxisomal membrane and is essential for efficient peroxisomal targeting of proteins containing peroxisomal targeting signal 1. MoPEX19 deletion leads to the cytoplasmic distribution of Mopex14/17, indicating that the peroxisomal import of Pex14/17 is dependent on Pex19. The knockout mutants of MoPEX14/17 show reduced fatty acid utilization, reactive oxygen species (ROS) degradation and cell wall integrity. Moreover, Δmopex14/17 mutants show delayed conidial generation and appressorial formation, and a reduction in appressorial turgor accumulation and penetration ability in host plants. These defects result in a significant reduction in the virulence of the mutant. These data indicate that MoPEX14/17 plays a crucial role in peroxisome biogenesis and contributes to fungal development and pathogenicity.     

Peroxisome fission is associated with reorganization of specific membrane proteins

Membrane remodeling is an important aspect in organelle biogenesis. We show that different peroxisome membrane proteins that play a role in organelle biogenesis and proliferation (Pex8, Pex10, Pex14, Pex25 and Pex11) are subject to spatiotemporal behavior during organelle development. Using fluorescence microscopy analysis of Hansenula polymorpha dnm1 cells that are blocked in the normal fission process, we show that green fluorescent protein (GFP) fusions of Pex8, Pex10, Pex14 and Pex25 show enhanced fluorescence at the organelle extensions that are formed in budding cells. In contrast, Pex11 fluorescence is enriched at the base of this extension on the mother organelle. A fusion protein of GFP with the transporter Pmp47, used as a control, did not show enhanced fluorescence at any specific region of the organelle. The concentration of specific peroxins at the peroxisome surface was lost upon deletion of PEX11 or the N-terminal domain of Pex11 that is involved in membrane remodeling. Comparable distribution patterns as in dnm1 cells were observed in wild-type cells where Pex8, Pex10, Pex14 and Pex25, but not Pex11, were especially present at newly formed organelles that migrated to the bud. We speculate that peroxin reorganization events result in enhanced levels of peroxins involved in peroxisome biogenesis in nascent organelles.     

The human PEX3 gene encoding a peroxisomal assembly protein: genomic organization, positional mapping, and mutation analysis in candidate phenotypes

In yeasts, the peroxin Pex3p was identified as a peroxisomal integral membrane protein that presumably plays a role in the early steps of peroxisomal assembly. In humans, defects of peroxins cause peroxisomal biogenesis disorders such as Zellweger syndrome. We previously reported data on the human PEX3 cDNA and its protein, which in addition to the peroxisomal targeting sequence contains a putative endoplasmic reticulum targeting signal. Here we report the genomic organization, sequencing of the putative promoter region, chromosomal localization, and physical mapping of the human PEX3 gene. The gene is composed of 12 exons and 11 introns spanning a region of approximately 40 kb. The highly conserved putative promoter region is very GC rich, lacks typical TATA and CCAAT boxes, and contains potential Sp1, AP1, and AP2 binding sites. The gene was localized to chromosome 6q23-24 and D6S279 was identified to be the closest positional marker. As yeast mutants deficient in PEX3 have been shown to lack peroxisomes as well as any peroxisomal remnant structures, human PEX3 is a candidate gene for peroxisomal assembly disorders. Mutation analysis of the human PEX3 gene was therefore performed in fibroblasts from patients suffering from peroxisome biogenesis disorders. Complementation groups 1, 4, 7, 8, and 9 according to the numbering system of Kennedy Krieger Institute were analyzed but no difference to the wild-type sequence was detected. PEX3 mutations were therefore excluded as the molecular basis of the peroxisomal defect in these complementation groups.     

Genomic organization, expression analysis, and chromosomal localization of the mouse PEX3 gene encoding a peroxisomal assembly protein

The peroxin Pex3p has been identified as an integral peroxisomal membrane protein in yeast where pex3 mutants lack peroxisomal remnant structures. Although not proven in higher organisms, a role of this gene in the early peroxisome biogenesis is suggested. We report here the cDNA cloning and the genomic structure of the mouse PEX3 gene. The 2 kb cDNA encodes a polypeptide of 372 amino acids (42 kDa). The gene spans a region of 30 kb, contains 12 exons and 11 introns and is located on band A of chromosome 10. The putative promoter region exhibits characteristic housekeeping features. PEX3 expression was identified in all tissues analyzed, with the strongest signals in liver and in testis, and could not be induced by fenofibrate. The data presented may be useful for the generation of a mouse model defective in PEX3 in order to clarify the yet unknown functional impact of disturbances in early peroxisomal membrane assembly.     

Pex7p and Pex20p of Neurospora crassa function together in PTS2-dependent protein import into peroxisomes

Recruiting matrix proteins with a peroxisomal targeting signal type 2 (PTS2) to the peroxisomal membrane requires species-specific factors. In Saccharomyces cerevisiae, the PTS2 receptor Pex7p acts in concert with the redundant Pex18p/Pex21p, whereas in Yarrowia lipolytica, Pex20p might unite the function of both S. cerevisiae peroxins. Herein, the genome of the filamentous fungus Neurospora crassa was analyzed for peroxin-encoding genes. We identified a set of 18 peroxins that resembles that of Y. lipolytica rather than that of S. cerevisiae. Interestingly, proteins homologous to both S. cerevisiae Pex7p and Y. lipolytica Pex20p exist in N. crassa. We report on the isolation of these PTS2-specific peroxins and demonstrate that NcPex20p can substitute for S. cerevisiae Pex18p/Pex21p, but not for ScPex7p. Like Pex18p, NcPex20p did not bind PTS2 protein or the docking proteins in the absence of ScPex7p. Rather, NcPex20p was required before docking to form an import-competent complex of cargo-loaded PTS2 receptors. NcPex7p did not functionally replace yeast Pex7p, probably because the N. crassa PTS2 receptor failed to associate with Pex18p/Pex21p. However, once NcPex7p and NcPex20p had been coexpressed, it proved possible to replace yeast Pex7p. Pex20p and Pex18p/Pex21p are therefore true orthologues, both of which are in need of Pex7p for PTS2 protein import.     

Mutation in PEX16 is causal in the peroxisome-deficient Zellweger syndrome of complementation group D.

Peroxisome-biogenesis disorders (PBDs), including Zellweger syndrome (ZS), are autosomal recessive diseases caused by a deficiency in peroxisome assembly as well as by a malfunction of peroxisomes, among which>10 genotypes have been identified. We have isolated a human PEX16 cDNA (HsPEX16) by performing an expressed-sequence-tag homology search on a human DNA database, by using yeast PEX16 from Yarrowia lipolytica and then screening the human liver cDNA library. This cDNA encodes a peroxisomal protein (a peroxin Pex16p) made up of 336 amino acids. Among 13 peroxisome-deficiency complementation groups (CGs), HsPEX16 expression morphologically and biochemically restored peroxisome biogenesis only in fibroblasts from a CG-D patient with ZS in Japan (the same group as CG-IX in the United States). Pex16p was localized to peroxisomes through expression study of epitope-tagged Pex16p. One patient (PBDD-01) possessed a homozygous, inactivating nonsense mutation, C-->T at position 526 in a codon (CGA) for 176Arg, that resulted in a termination codon (TGA). This implies that the C-terminal half is required for the biological function of Pex16p. PBDD-01-derived PEX16 cDNA was defective in peroxisome-restoring activity when expressed in the patient's fibroblasts. These results demonstrate that mutation in PEX16 is the genetic cause of CG-D PBDs.     

Pexophagy is responsible for 65% of cases of peroxisome biogenesis disorders

Peroxisome biogenesis disorders (PBDs) is a group of diseases caused by mutations in one of the peroxins, proteins responsible for biogenesis of the peroxisomes. In recent years, it became clear that many peroxins (e.g., PEX3 and PEX14) play additional roles in peroxisome homeostasis (such as promoting autophagic degradation of peroxisomes or pexophagy), which are often opposite to their originally established functions in peroxisome formation and maintenance. Even more interesting, the peroxins that make up the peroxisomal AAA ATPase complex (AAA-complex) in yeast (Pex1, Pex6 and Pex15) or mammals (PEX1, PEX6, PEX26) are responsible for the downregulation of pexophagy. Moreover, this might be even their primary role in human: to prevent pexophagy by removing from the peroxisomal membrane the ubiquitinated peroxisomal matrix protein import receptor, Ub-PEX5, which is also a signal for the Ub-binding pexophagy receptor, NBR1. Remarkably, the peroxisomes rescued from pexophagy by autophagic inhibitors in PEX1^G843D (the most common PBD mutation) cells are able to import matrix proteins and improve their biochemical function suggesting that the AAA-complex per se is not essential for the protein import function in human. This paradigm-shifting discovery published in the current issue of Autophagy has raised hope for up to 65% of all PBD patients with various deficiencies in the AAA-complex. Recognizing PEX1, PEX6 and PEX26 as pexophagy suppressors will allow treating these patients with a new range of tools designed to target mammalian pexophagy.     

Pex15p of Saccharomyces cerevisiae provides a molecular basis for recruitment of the AAA peroxin Pex6p to peroxisomal membranes

The gene products (peroxins) of at least 29 PEX genes are known to be necessary for peroxisome biogenesis but for most of them their precise function remains to be established. Here we show that Pex15p, an integral peroxisomal membrane protein, in vivo and in vitro binds the AAA peroxin Pex6p. This interaction functionally interconnects these two hitherto unrelated peroxins. Pex15p provides the mechanistic basis for the reversible targeting of Pex6p to peroxisomal membranes. We could demonstrate that the N-terminal part of Pex6p contains the binding site for Pex15p and that the two AAA cassettes D1 and D2 of Pex6p have opposite effects on this interaction. A point mutation in the Walker A motif of D1 (K489A) decreased the binding of Pex6p to Pex15p indicating that the interaction of Pex6p with Pex15p required binding of ATP. Mutations in Walker A (K778A) and B (D831Q) motifs of D2 abolished growth on oleate and led to a considerable larger fraction of peroxisome bound Pex6p. The nature of these mutations suggested that ATP-hydrolysis is required to disconnect Pex6p from Pex15p. On the basis of these results, we propose that Pex6p exerts at least part of its function by an ATP-dependent cycle of recruitment and release to and from Pex15p.     

Peroxisome biogenesis disorders: Molecular basis for impaired peroxisomal membrane assembly: In metabolic functions and biogenesis of peroxisomes in health and disease

Peroxisome is a single-membrane organelle in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome (ZS). Gene defects of peroxins required for both membrane assembly and matrix protein import are identified: ten mammalian pathogenic peroxins for ten complementation groups of PBDs, are required for matrix protein import; three, Pex3p, Pex16p and Pex19p, are shown to be essential for peroxisome membrane assembly and responsible for the most severe ZS in PBDs of three complementation groups 12, 9, and 14, respectively. Patients with severe ZS with defects of PEX3, PEX16, and PEX19 tend to carry severe mutation such as nonsense mutations, frameshifts and deletions. With respect to the function of these three peroxins in membrane biogenesis, two distinct pathways have been proposed for the import of peroxisomal membrane proteins in mammalian cells: a Pex19p- and Pex3p-dependent class I pathway and a Pex19p- and Pex16p-dependent class II pathway. In class II pathway, Pex19p also forms a soluble complex with newly synthesized Pex3p as the chaperone for Pex3p in the cytosol and directly translocates it to peroxisomes. Pex16p functions as the peroxisomal membrane receptor that is specific to the Pex3p-Pex19p complexes. A model for the import of peroxisomal membrane proteins is suggested, providing new insights into the molecular mechanisms underlying the biogenesis of peroxisomes and its regulation involving Pex3p, Pex19p, and Pex16p. Another model suggests that in Saccharomyces cerevisiae peroxisomes likely emerge from the endoplasmic reticulum. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of peroxisomes in Health and Disease.     

Pex30p, Pex31p, and Pex32p form a family of peroxisomal integral membrane proteins regulating peroxisome size and number in Saccharomyces cerevisiae

The peroxin Pex23p of the yeast Yarrowia lipolytica exhibits high sequence similarity to the hypothetical proteins Ylr324p, Ygr004p, and Ybr168p encoded by the Saccharomyces cerevisiae genome. Ylr324p, Ygr004p, and Ybr168p are integral to the peroxisomal membrane and act to control peroxisome number and size. Synthesis of Ylr324p and Ybr168p, but not of Ygr004p, is induced during incubation of cells in oleic acid-containing medium, the metabolism of which requires intact peroxisomes. Cells deleted for YLR324w exhibit increased numbers of peroxisomes, whereas cells deleted for YGR004w or YBR168w exhibit enlarged peroxisomes. Ylr324p and Ybr168p cannot functionally substitute for one another or for Ygr004p, whereas Ygr004p shows partial functional redundancy with Ylr324p and Ybr168p. Ylr324p, Ygr004p, and Ybr168p interact within themselves and with Pex28p and Pex29p, which have been shown also to regulate peroxisome size and number. Systematic deletion of genes demonstrated that PEX28 and PEX29 function upstream of YLR324w, YGR004w, and YBR168w in the regulation of peroxisome proliferation. Our data suggest a role for Ylr324p, Ygr004p, and Ybr168p--now designated Pex30p, Pex31p, and Pex32p, respectively--together with Pex28p and Pex29p in controlling peroxisome size and proliferation in Saccharomyces cerevisiae.