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.     

Dysferlin domain-containing proteins, Pex30p and Pex31p, localized to two compartments, control the number and size of oleate-induced peroxisomes in Pichia pastoris

Yarrowia lipolytica Pex23p and Saccharomyces cerevisiae Pex30p, Pex31p, and Pex32p comprise a family of dysferlin domain-containing peroxins. We show that the deletion of their Pichia pastoris homologues, PEX30 and PEX31, does not affect the function or division of methanol-induced peroxisomes but results in fewer and enlarged, functional, oleate-induced peroxisomes. Synthesis of Pex30p is constitutive, whereas that of Pex31p is oleate-induced but at a much lower level relative to Pex30p. Pex30p interacts with Pex31p and is required for its stability. At steady state, both Pex30p and Pex31p exhibit a dual localization to the endoplasmic reticulum (ER) and peroxisomes. However, Pex30p is localized mostly to the ER, whereas Pex31p is predominantly on peroxisomes. Consistent with ER-to-peroxisome trafficking of these proteins, Pex30p accumulates on peroxisomes upon overexpression of Pex31p. Additionally, Pex31p colocalizes with Pex30p at the ER in pex19Delta cells and can be chased from the ER to peroxisomes in a Pex19p-dependent manner. The dysferlin domains of Pex30p and Pex31p, which are dispensable for their interaction, stability, and subcellular localization, are essential for normal peroxisome number and size. The growth environment-specific role of these peroxins, their dual localization, and the function of their dysferlin domains provide novel insights into peroxisome morphogenesis.     

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.     

Pex11p plays a primary role in medium-chain fatty acid oxidation, a process that affects peroxisome number and size in Saccharomyces cerevisiae

The Saccharomyces cerevisiae peroxisomal membrane protein Pex11p has previously been implicated in peroxisome proliferation based on morphological observations of PEX11 mutant cells. Pex11p-deficient cells fail to increase peroxisome number in response to growth on fatty acids and instead accumulate a few giant peroxisomes. We report that mutants deficient in genes required for medium-chain fatty acid (MCFA) beta-oxidation display the same phenotype as Pex11p-deficient cells. Upon closer inspection, we found that Pex11p is required for MCFA beta-oxidation. Disruption of the PEX11 gene results in impaired formation of MCFA-CoA esters as measured in intact cells, whereas their formation is normal in cell lysates. The sole S. cerevisiae MCFA-CoA synthetase (Faa2p) remains properly localized to the inner leaflet of the peroxisomal membrane in PEX11 mutant cells. Therefore, the in vivo latency of MCFA activation observed in Pex11p-deficient cells suggests that Pex11p provides Faa2p with substrate. When PEX11 mutant cells are shifted from glucose to oleate-containing medium, we observed an immediate deficiency in beta-oxidation of MCFAs whereas giant peroxisomes and a failure to increase peroxisome abundance only became apparent much later. Our observations suggest that the MCFA oxidation pathway regulates the level of a signaling molecule that modulates the number of peroxisomal structures in a cell.     

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

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

Pex3p initiates the formation of a preperoxisomal compartment from a subdomain of the endoplasmic reticulum in Saccharomyces cerevisiae

Peroxisomes are dynamic organelles that often proliferate in response to compounds that they metabolize. Peroxisomes can proliferate by two apparent mechanisms, division of preexisting peroxisomes and de novo synthesis of peroxisomes. Evidence for de novo peroxisome synthesis comes from studies of cells lacking the peroxisomal integral membrane peroxin Pex3p. These cells lack peroxisomes, but peroxisomes can assemble upon reintroduction of Pex3p. The source of these peroxisomes has been the subject of debate. Here, we show that the amino-terminal 46 amino acids of Pex3p of Saccharomyces cerevisiae target to a subdomain of the endoplasmic reticulum and initiate the formation of a preperoxisomal compartment for de novo peroxisome synthesis. In vivo video microscopy showed that this preperoxisomal compartment can import both peroxisomal matrix and membrane proteins leading to the formation of bona fide peroxisomes through the continued activity of full-length Pex3p. Peroxisome formation from the preperoxisomal compartment depends on the activity of the genes PEX14 and PEX19, which are required for the targeting of peroxisomal matrix and membrane proteins, respectively. Our findings support a direct role for the endoplasmic reticulum in de novo peroxisome formation.     

Pex19p Interacts with Pex3p and Pex10p and Is Essential for Peroxisome Biogenesis in Pichia pastoris

We report the cloning and characterization of Pichia pastoris PEX19 by complementation of a peroxisome-deficient mutant strain. Import of peroxisomal targeting signal 1- and 2-containing peroxisomal matrix proteins is defective in pex19 mutants. PEX19 encodes a hydrophilic 299-amino acid protein with sequence similarity to Saccharomyces cerevisiae Pex19p and human and Chinese hamster PxF, all farnesylated proteins, as well as hypothetical proteins from Caenorhabditis elegans and Schizosaccharomyces pombe. The farnesylation consensus is conserved in PpPex19p but dispensable for function and appears unmodified under the conditions tested. Pex19p localizes predominantly to the cytosolic fraction. Biochemical and two-hybrid analyses confirmed that Pex19p interacts with Pex3p, as seen in S. cerevisiae, but unexpectedly also with Pex10p. Two-hybrid analysis demonstrated that the amino-terminal 42 amino acids of Pex19p interact with the carboxyl-terminal 335 amino acids of Pex3p. In addition, the extreme carboxyl terminus of Pex19p (67 amino acids) is required for interaction with the amino-terminal 380 amino acids of Pex10p. Biochemical and immunofluorescence microscopy analyses of pex19Delta cells identified the membrane protein Pex3p in peroxisome remnants that were not previously observed in S. cerevisiae. These small vesicular and tubular (early) remnants are morphologically distinct from other Pppex mutant (late) remnants, suggesting that Pex19p functions at an early stage of 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.     

Pex19p binds Pex30p and Pex32p at regions required for their peroxisomal localization but separate from their peroxisomal targeting signals

The assembly of proteins in the peroxisomal membrane is a multistep process requiring their recognition in the cytosol, targeting to and insertion into the peroxisomal membrane, and stabilization within the lipid bilayer. The peroxin Pex19p has been proposed to be either the receptor that recognizes and targets newly synthesized peroxisomal membrane proteins (PMP) to the peroxisome or a chaperone required for stabilization of PMPs at the peroxisomal membrane. Differentiating between these two roles for Pex19p could be achieved by determining whether the peroxisomal targeting signal (PTS) and the region of Pex19p binding of a PMP are the same or different. We addressed the role for Pex19p in the assembly of two PMPs, Pex30p and Pex32p, of the yeast Saccharomyces cerevisiae. Pex30p and Pex32p control peroxisome size and number but are dispensable for peroxisome formation. Systematic truncations from the carboxyl terminus, together with in-frame deletions of specific regions, have identified PTSs essential for targeting Pex30p and Pex32p to peroxisomes. Both Pex30p and Pex32p interact with Pex19p in regions that do not overlap with their PTSs. However, Pex19p is required for localizing Pex30p and Pex32p to peroxisomes, because mutations that disrupt the interaction of Pex19p with Pex30p and Pex32p lead to their mislocalization to a compartment other than peroxisomes. Mutants of Pex30p and Pex32p that localize to peroxisomes but produce cells exhibiting the peroxisomal phenotypes of cells lacking these proteins demonstrate that the regions in these proteins that control peroxisomal targeting and cell biological activity are separable. Together, our data show that the interaction of Pex19p with Pex30p and Pex32p is required for their roles in peroxisome biogenesis and are consistent with a chaperone role for Pex19p in stabilizing or maintaining membrane proteins in peroxisomes.     

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.     

A dual function for Pex3p in peroxisome formation and inheritance

Saccharomyces cerevisiae Pex3p has been shown to act at the ER during de novo peroxisome formation. However, its steady state is at the peroxisomal membrane, where its role is debated. Here we show that Pex3p has a dual function: one in peroxisome formation and one in peroxisome segregation. We show that the peroxisome retention factor Inp1p interacts physically with Pex3p in vitro and in vivo, and split-GFP analysis shows that the site of interaction is the peroxisomal membrane. Furthermore, we have generated PEX3 alleles that support peroxisome formation but fail to support recruitment of Inp1p to peroxisomes, and as a consequence are affected in peroxisome segregation. We conclude that Pex3p functions as an anchor for Inp1p at the peroxisomal membrane, and that this function is independent of its role at the ER in peroxisome biogenesis.     

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.     

Domain mapping of human PEX5 reveals functional and structural similarities to Saccharomyces cerevisiae Pex18p and Pex21p

PEX5 functions as an import receptor for proteins with the type-1 peroxisomal targeting signal (PTS1). Although PEX5 is not involved in the import of PTS2-targeted proteins in yeast, it is essential for PTS2 protein import in mammalian cells. Human cells generate two isoforms of PEX5 through alternative splicing, PEX5S and PEX5L, and PEX5L contains an additional insert 37 amino acids long. Only one isoform, PEX5L, is involved in PTS2 protein import, and PEX5L physically interacts with PEX7, the import receptor for PTS2-containing proteins. In this report we map the regions of human PEX5L involved in PTS2 protein import, PEX7 interaction, and targeting to peroxisomes. These studies revealed that amino acids 1-230 of PEX5L are required for PTS2 protein import, amino acids 191-222 are sufficient for PEX7 interaction, and amino acids 1-214 are sufficient for targeting to peroxisomes. We also identified a 21-amino acid-long peptide motif of PEX5L, amino acids 209-229, that overlaps the regions sufficient for full PTS2 rescue activity and PEX7 interaction and is shared by Saccharomyces cerevisiae Pex18p and Pex21p, two yeast peroxins that act only in PTS2 protein import in yeast. A mutation in PEX5 that changes a conserved serine of this motif abrogates PTS2 protein import in mammalian cells and reduces the interaction of PEX5L and PEX7 in vitro. This peptide motif also lies within regions of Pex18p and Pex21p that interact with yeast PEX7. Based on these and other results, we propose that mammalian PEX5L may have acquired some of the functions that yeast Pex18p and/or Pex21p perform in PTS2 protein import. This hypothesis may explain the essential role of PEX5L in PTS2 protein import in mammalian cells and its lack of importance for PTS2 protein import in yeast.     

Pex7p translocates in and out of peroxisomes in Saccharomyces cerevisiae

Pex7p is the soluble receptor responsible for importing into peroxisomes newly synthesized proteins bearing a type 2 peroxisomal targeting sequence. We observe that appending GFP to Pex7p's COOH terminus shifts Pex7p's intracellular distribution from predominantly cytosolic to predominantly peroxisomal in Saccharomyces cerevisiae. Cleavage of the link between Pex7p and GFP within peroxisomes liberates GFP, which remains inside the organelle, and Pex7p, which exits to the cytosol. The reexported Pex7p is functional, resulting in import of thiolase into peroxisomes and improved growth of the yeast on oleic acid. These results support the "extended shuttle" model of peroxisome import receptor function and open the way to future studies of receptor export.     

The dynamin-like protein Vps1p of the yeast Saccharomyces cerevisiae associates with peroxisomes in a Pex19p-dependent manner

Dynamins and dynamin-like proteins play important roles in organelle division. In Saccharomyces cerevisiae, the dynamin-like protein Vps1p (vacuolar protein sorting protein 1) is involved in peroxisome fission, as cells deleted for the VPS1 gene contain reduced numbers of enlarged peroxisomes. What relationship Vps1p has with peroxisomes remains unclear. Here we show that Vps1p interacts with Pex19p, a peroxin that acts as a shuttling receptor for peroxisomal membrane proteins or as a chaperone assisting the assembly/stabilization of proteins at the peroxisome membrane. Vps1p contains two putative Pex19p recognition sequences at amino acids 509-523 and 633-647. Deletion of the first (but not the second) sequence results in reduced numbers of enlarged peroxisomes in cells, as in vps1delta cells. Deletion of either sequence has no effect on vacuolar morphology or vacuolar protein sorting, suggesting that the peroxisome and vacuole biogenic functions of Vps1p are separate and separable. Substitution of proline for valine at position 516 of Vps1p abrogates Pex19p binding and gives the peroxisome phenotype of vps1delta cells. Microscopic analysis showed that overexpression of Pex19p or redirection of Pex19p to the nucleus does not affect the normal cellular distribution of Vps1p in the cytosol and in punctate structures that are not peroxisomes, suggesting that Pex19p does not function in targeting Vps1p to peroxisomes. Subcellular fractionation showed that a fraction of Vps1p is associated with peroxisomes and that deletion or mutation of the first Pex19p recognition sequence abrogates this association. Our results are consistent with Pex19p acting as a chaperone to stabilize the association of Vps1p with peroxisomes and not as a receptor involved in targeting Vps1p to peroxisomes.     

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.     

Mutations in the peroxin Pex26p responsible for peroxisome biogenesis disorders of complementation group 8 impair its stability, peroxisomal localization, and interaction with the Pex1p x Pex6p complex

Peroxisome biogenesis disorders (PBDs) are fatal autosomal recessive diseases and are caused by impaired peroxisome biogenesis. PBDs are genetically heterogeneous and classified into 13 complementation groups (CGs). CG8 is one of the most common groups and has three clinical phenotypes, including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy, and infantile Refsum disease (IRD). We recently isolated PEX26 as the pathogenic gene for PBD of CG8. Pex26p functions in recruiting to peroxisomes the complexes of the AAA ATPase peroxins, Pex1p and Pex6p. In the present work, we identified four distinct mutations in PEX26 from five patients of CG8 PBD including 2 with ZS and 3 with IRD, in addition to 7 mutant alleles in 8 patients in the first report describing the pathogenic PEX26 gene for CG8 PBD. Phenotype-genotype analyses revealed that temperature-sensitive (ts) peroxisome assembly gave rise to a milder IRD in contrast to the non-ts phenotype of the cells from ZS patients. Furthermore, we present several lines of evidence that show that the instability, insufficient binding to Pex1p x Pex6p complexes, or mislocalization of patient-derived Pex26p mutants is most likely responsible for the CG8 PBDs.     

The peroxin Pex34p functions with the Pex11 family of peroxisomal divisional proteins to regulate the peroxisome population in yeast

Peroxisomes are ubiquitous organelles involved in diverse metabolic processes, most notably the metabolism of lipids and the detoxification of reactive oxygen species. Peroxisomes are highly dynamic and change in size and number in response to both intra- and extracellular cues. In the yeast Saccharomyces cerevisiae, peroxisome growth and division are controlled by both the differential import of soluble matrix proteins and a specialized divisional machinery that includes peroxisome-specific factors, such as members of the Pex11 protein family, and general organelle divisional factors, such as the dynamin-related protein Vps1p. Global yeast two-hybrid analyses have demonstrated interactions between the product of the S. cerevisiae gene of unknown function, YCL056c, and Pex proteins involved in peroxisome biogenesis. Here we show that the protein encoded by YCL056c, renamed Pex34p, is a peroxisomal integral membrane protein that acts independently and also in concert with the Pex11 protein family members Pex11p, Pex25p, and Pex27p to control the peroxisome populations of cells under conditions of both peroxisome proliferation and constitutive peroxisome division. Yeast two-hybrid analysis showed that Pex34p interacts physically with itself and with Pex11p, Pex25p, and Pex27p but not with Vps1p. Pex34p can act as a positive effector of peroxisome division as its overexpression leads to increased numbers of peroxisomes in wild type and pex34Δ cells. Pex34p requires the Pex11 family proteins to promote peroxisome division. Our discovery of Pex34p as a protein involved in the already complex control of peroxisome populations emphasizes the necessity of cells to strictly regulate their peroxisome populations to be able to respond appropriately to changing environmental conditions.