Pex20p functions as the receptor for non‐PTS1/non‐PTS2 acyl‐CoA oxidase import into peroxisomes of the yeast Yarrowia lipolytica

Most soluble proteins targeted to the peroxisomal matrix contain a C‐terminal peroxisome targeting signal type 1 (PTS1) or an N‐terminal PTS2 that is recognized by the receptors Pex5p and Pex7p, respectively. These receptors cycle between the cytosol and peroxisome and back again for multiple rounds of cargo delivery to the peroxisome. A small number of peroxisomal matrix proteins, including all six isozymes of peroxisomal fatty acyl‐CoA oxidase (Aox) of the yeast Yarrowia lipolytica, contain neither a PTS1 nor a PTS2. Pex20p has been shown to function as a co‐receptor for Pex7p in the import of PTS2 cargo into peroxisomes. Here we show that cells of Y. lipolytica deleted for the PEX20 gene fail to import not only the PTS2‐containing protein 3‐ketoacyl‐CoA thiolase (Pot1p) but also the non‐PTS1/non‐PTS2 Aox isozymes. Pex20p binds directly to Aox isozymes Aox3p and Aox5p, which requires the C‐terminal Wxxx(F/Y) motif of Pex20p. A W411G mutation in the C‐terminal Wxxx(F/Y) motif causes Aox isozymes to be mislocalized to the cytosol. Pex20p interacts physically with members of the peroxisomal import docking complex, Pex13p and Pex14p. Our results are consistent with a role for Pex20p as the receptor for import of the non‐PTS1/non‐PTS2 Aox isozymes into peroxisomes.     

A signal from inside the peroxisome initiates its division by promoting the remodeling of the peroxisomal membrane

We define the dynamics of spatial and temporal reorganization of the team of proteins and lipids serving peroxisome division. The peroxisome becomes competent for division only after it acquires the complete set of matrix proteins involved in lipid metabolism. Overloading the peroxisome with matrix proteins promotes the relocation of acyl-CoA oxidase (Aox), an enzyme of fatty acid beta-oxidation, from the matrix to the membrane. The binding of Aox to Pex16p, a membrane-associated peroxin required for peroxisome biogenesis, initiates the biosynthesis of phosphatidic acid and diacylglycerol (DAG) in the membrane. The formation of these two lipids and the subsequent transbilayer movement of DAG initiate the assembly of a complex between the peroxins Pex10p and Pex19p, the dynamin-like GTPase Vps1p, and several actin cytoskeletal proteins on the peroxisomal surface. This protein team promotes membrane fission, thereby executing the terminal step of peroxisome division.     

Involvement of the endoplasmic reticulum in peroxisome formation

The traditional view holds that peroxisomes are autonomous organelles multiplying by growth and division. More recently, new observations have challenged this concept. Herein, we present evidence supporting the involvement of the endoplasmic reticulum (ER) in peroxisome formation by electron microscopy, immunocytochemistry and three-dimensional image reconstruction of peroxisomes and associated compartments in mouse dendritic cells. We found the peroxisomal membrane protein Pex13p and the ATP-binding cassette transporter protein PMP70 present in specialized subdomains of the ER that were continuous with a peroxisomal reticulum from which mature peroxisomes arose. The matrix proteins catalase and thiolase were only detectable in the reticula and peroxisomes. Our results suggest the existence of a maturation pathway from the ER to peroxisomes and implicate the ER as a major source from which the peroxisomal membrane is derived.     

Acyl-CoA oxidase is imported as a heteropentameric, cofactor-containing complex into peroxisomes of Yarrowia lipolytica

Five isoforms of acyl-CoA oxidase (Aox), designated Aox1p to Aox5p, constitute a 443-kD heteropentameric complex containing one polypeptide chain of each isoform within the peroxisomal matrix of the yeast Yarrowia lipolytica. Assembly of the Aox complex occurs in the cytosol and precedes its import into peroxisomes. Peroxisomal targeting of the Aox complex is abolished in a mutant lacking the peroxin Pex5p, a component of the matrix protein targeting machinery. Import of the Aox complex into peroxisomes does not involve the cytosolic chaperone Pex20p, which mediates the oligomerization and import of peroxisomal thiolase. Aox2p and Aox3p play a pivotal role in the formation of the Aox complex in the cytosol and can substitute for one another in promoting assembly of the complex. In vitro, these subunits retard disassembly of the Aox complex and increase the efficiency of its reassembly. Neither Aox2p nor Aox3p is required for acquisition of the cofactor FAD by other components of the complex. We provide evidence that the Aox2p- and Aox3p-assisted assembly of the Aox complex in the cytosol is mandatory for its import into peroxisomes and that no component of the complex can penetrate the peroxisomal matrix as a monomer.     

Dynamic ergosterol- and ceramide-rich domains in the peroxisomal membrane serve as an organizing platform for peroxisome fusion

We describe unusual ergosterol- and ceramide-rich (ECR) domains in the membrane of yeast peroxisomes. Several key features of these detergent-resistant domains, including the nature of their sphingolipid constituent and its unusual distribution across the membrane bilayer, clearly distinguish them from well characterized detergent-insoluble lipid rafts in the plasma membrane. A distinct set of peroxisomal proteins, including two ATPases, Pex1p and Pex6p, as well as phosphoinositide- and GTP-binding proteins, transiently associates with the cytosolic face of ECR domains. All of these proteins are essential for the fusion of the immature peroxisomal vesicles P1 and P2, the earliest intermediates in a multistep pathway leading to the formation of mature, metabolically active peroxisomes. Peroxisome fusion depends on the lateral movement of Pex1p, Pex6p, and phosphatidylinositol-4,5-bisphosphate-binding proteins from ECR domains to a detergent-soluble portion of the membrane, followed by their release to the cytosol. Our data suggest a model for the multistep reorganization of the multicomponent peroxisome fusion machinery that transiently associates with ECR domains.     

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.     

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.     

Peroxisome biogenesis occurs in an unsynchronized manner in close association with the endoplasmic reticulum in temperature-sensitive Yarrowia lipolytica Pex3p mutants

Pex3p is a peroxisomal integral membrane protein required early in peroxisome biogenesis, and Pex3p-deficient cells lack identifiable peroxisomes. Two temperature-sensitive pex3 mutant strains of the yeast Yarrowia lipolytica were made to investigate the role of Pex3p in the early stages of peroxisome biogenesis. In glucose medium at 16 degrees C, these mutants underwent de novo peroxisome biogenesis and exhibited early matrix protein sequestration into peroxisome-like structures found at the endoplasmic reticulum-rich periphery of cells or sometimes associated with nuclei. The de novo peroxisome biogenesis seemed unsynchronized, with peroxisomes occurring at different stages of development both within cells and between cells. Cells with peripheral nascent peroxisomes and cells with structures morphologically distinct from peroxisomes, such as semi/circular tubular structures that immunostained with antibodies to peroxisomal matrix proteins and to the endoplasmic reticulum-resident protein Kar2p, and that surrounded lipid droplets, were observed during up-regulation of peroxisome biogenesis in cells incubated in oleic acid medium at 16 degrees C. These structures were not detected in wild-type or Pex3p-deficient cells. Their role in peroxisome biogenesis remains unclear. Targeting of peroxisomal matrix proteins to these structures suggests that Pex3p directly or indirectly sequesters components of the peroxisome biogenesis machinery. Such a role is consistent with Pex3p overexpression producing cells with fewer, larger, and clustered peroxisomes.     

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.     

Localization of p21-Activated Kinase 1 (PAK1) to Pinocytic Vesicles and Cortical Actin Structures in Stimulated Cells

Pex mutants of the yeast Yarrowia lipolytica are defective in peroxisome assembly. The mutant strain pex16-1 lacks morphologically recognizable peroxisomes. Most peroxisomal proteins are mislocalized to a subcellular fraction enriched for cytosol in pex16 strains, but a subset of peroxisomal proteins is localized at, or near, wild-type levels to a fraction typically enriched for peroxisomes. The PEX16 gene was isolated by functional complementation of the pex16-1 strain and encodes a protein, Pex16p, of 391 amino acids (44,479 D). Pex16p has no known homologues. Pex16p is a peripheral protein located at the matrix face of the peroxisomal membrane. Substitution of the carboxylterminal tripeptide Ser-Thr-Leu, which is similar to the consensus sequence of peroxisomal targeting signal 1, does not affect targeting of Pex16p to peroxisomes. Pex16p is synthesized in wild-type cells grown in glucose-containing media, and its levels are modestly increased by growth of cells in oleic acid–containing medium. Overexpression of the PEX16 gene in oleic acid– grown Y. lipolytica leads to the appearance of a small number of enlarged peroxisomes, which contain the normal complement of peroxisomal proteins at levels approaching those of wild-type peroxisomes.     

Sorting pathway and molecular targeting signals for the Arabidopsis peroxin 3

Peroxin 3 (Pex3p) has been identified and characterized as a peroxisomal membrane protein in yeasts and mammals. We identified two putative homologs in Arabidopsis (AtPex3p, forms 1 and 2), both with an identical cluster of positively charged amino acid residues (RKHRRK) immediately preceding one of the two predicted transmembrane domains (TMD1). In transiently transformed Arabidopsis and tobacco BY-2 suspension-cultured cells, epitope-tagged AtPex3p (form 2) sorted post-translationally from the cytosol directly to peroxisomes, the first sorting pathway described for any peroxin in plants. TMD1 and RKHRRK were necessary for targeting form 2 to peroxisomes and sufficient for directing chloramphenicol acetyltransferase to peroxisomes in both cell types. The N and C termini of AtPex3p (form 2) extend into the peroxisomal matrix, different from mammal and yeast Pex3 proteins. Thus, two authentic peroxisomal membrane-bound Pex3p homologs possessing a membrane peroxisomal targeting signal, the first one defined for a plant peroxin and for any Pex3p homolog, exist in plant cells.     

A subtle interplay between three Pex11 proteins shapes de novo formation and fission of peroxisomes

The organization of eukaryotic cells into membrane-bound compartments must be faithfully sustained for survival of the cell. A subtle equilibrium exists between the degradation and the proliferation of organelles. Commonly, proliferation is initiated by a membrane remodeling process. Here, we dissect the function of proteins driving organelle proliferation in the particular case of peroxisomes. These organelles are formed either through a growth and division process from existing peroxisomes or de novo from the endoplasmic reticulum (ER). Among the proteins involved in the biogenesis of peroxisomes, peroxins, members of the Pex11 protein family participate in peroxisomal membrane alterations. In the yeast Saccharomyces cerevisiae, the Pex11 family consists of three proteins, Pex11p, Pex25p and Pex27p. Here we demonstrate that yeast mutants lacking peroxisomes require the presence of Pex25p to regenerate this organelle de novo. We also provide evidence showing that Pex27p inhibits peroxisomal function and illustrate that Pex25p initiates elongation of the peroxisomal membrane. Our data establish that although structurally conserved each of the three Pex11 protein family members plays a distinct role. While ScPex11p promotes the proliferation of peroxisomes already present in the cell, ScPex25p initiates remodeling at the peroxisomal membrane and ScPex27p acts to counter this activity. In addition, we reveal that ScPex25p acts in concert with Pex3p in the initiation of de novo peroxisome biogenesis from the ER.     

Reevaluation of the role of Pex1 and dynamin-related proteins in peroxisome membrane biogenesis

A recent model for peroxisome biogenesis postulates that peroxisomes form de novo continuously in wild-type cells by heterotypic fusion of endoplasmic reticulum–derived vesicles containing distinct sets of peroxisomal membrane proteins. This model proposes a role in vesicle fusion for the Pex1/Pex6 complex, which has an established role in matrix protein import. The growth and division model proposes that peroxisomes derive from existing peroxisomes. We tested these models by reexamining the role of Pex1/Pex6 and dynamin-related proteins in peroxisome biogenesis. We found that induced depletion of Pex1 blocks the import of matrix proteins but does not affect membrane protein delivery to peroxisomes; markers for the previously reported distinct vesicles colocalize in pex1 and pex6 cells; peroxisomes undergo continued growth if fission is blocked. Our data are compatible with the established primary role of the Pex1/Pex6 complex in matrix protein import and show that peroxisomes in Saccharomyces cerevisiae multiply mainly by growth and division.     

Identification of Pex5pM, and retarded maturation of 3-ketoacyl-CoA thiolase and acyl-CoA oxidase in CHO cells expressing mutant Pex5p isoforms

Recently, we isolated CHO cells, termed SK32 cells, that express mutant Pex5p (G432R), and showed mislocalization of catalase in the cytosol, but peroxisomal localization of 3-ketoacyl-CoA thiolase (thiolase) in the mutant cells [Ito, R. et al. (2001) Biochem. Biophys. Res. Commun. 288, 321-327]. While analyzing the mutant cells, we found a novel Pex5p isoform (Pex5pM), which was shorter by seven amino acids than Pex5pL and longer by 30 amino acids than Pex5pS. Similar levels of mRNA syntheses for the PEX5 gene were observed in both the wild type and mutant cells, but the protein levels of Pex5p isoforms were markedly reduced in the mutant cells cultured at 37 degrees C and only slightly discernible at 30 degrees C, suggesting that they could be rapidly degraded. Furthermore, we characterized the peroxisomal localization of thiolase and acyl-CoA oxidase (Aox) in SK32 cells. The proteins in the organelle fraction were protected from proteinase K-digestion in the mutant cells, indicating that they were translocated inside peroxisomes. However, the conversion of Aox from component A to components B and C was completely prevented at both 30 and 37 degrees C, and the precursor form of thiolase was partially processed to the mature one in a temperature-sensitive manner. Transformed SK32 cells stably expressing one of the wild type Pex5p isoforms were isolated, and then the maturation steps for thiolase and Aox were examined. Pex5pM and S restored the processing of the two enzymes, but Pex5pL did not. In addition, Pex5pL prevented the maturation of thiolase observed at 30 degrees C. These results indicate that (i) the novel Pex5pM is functional and (ii) a seven amino acids-insertion, which is present in the L isoform but absent in the M isoform, plays some role in the process of maturation of thiolase and Aox.     

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.     

Mutants of the Yarrowia lipolytica PEX23 gene encoding an integral peroxisomal membrane peroxin mislocalize matrix proteins and accumulate vesicles containing peroxisomal matrix and membrane proteins

pex mutants are defective in peroxisome assembly. The mutant strain pex23-1 of the yeast Yarrowia lipolytica lacks morphologically recognizable peroxisomes and mislocalizes all peroxisomal matrix proteins investigated preferentially to the cytosol. pex23 strains accumulate vesicular structures containing both peroxisomal matrix and membrane proteins. The PEX23 gene was isolated by functional complementation of the pex23-1 strain and encodes a protein, Pex23p, of 418 amino acids (47,588 Da). Pex23p exhibits high sequence similarity to two hypothetical proteins of the yeast Saccharomyces cerevisiae. Pex23p is an integral membrane protein of peroxisomes that is completely, or nearly completely, sequestered from the cytosol. Pex23p is detected at low levels in cells grown in medium containing glucose, and its levels are significantly increased by growth in medium containing oleic acid, the metabolism of which requires intact peroxisomes.     

The peroxisomal membrane protein import receptor Pex3p is directly transported to peroxisomes by a novel Pex19p- and Pex16p-dependent pathway

Two distinct pathways have recently been proposed for the import of peroxisomal membrane proteins (PMPs): a Pex19p- and Pex3p-dependent class I pathway and a Pex19p- and Pex3p-independent class II pathway. We show here that Pex19p plays an essential role as the chaperone for full-length Pex3p in the cytosol. Pex19p forms a soluble complex with newly synthesized Pex3p in the cytosol and directly translocates it to peroxisomes. Knockdown of Pex19p inhibits peroxisomal targeting of newly synthesized full-length Pex3p and results in failure of the peroxisomal localization of Pex3p. Moreover, we demonstrate that Pex16p functions as the Pex3p-docking site and serves as the peroxisomal membrane receptor that is specific to the Pex3p–Pex19p complexes. Based on these novel findings, we suggest a model for the import of PMPs that provides new insights into the molecular mechanisms underlying the biogenesis of peroxisomes and its regulation involving Pex3p, Pex19p, and Pex16p.     

Fusion of small peroxisomal vesicles in vitro reconstructs an early step in the in vivo multistep peroxisome assembly pathway of Yarrowia lipolytica

We have identified and purified six subforms of peroxisomes, designated P1 to P6, from the yeast, Yarrowia lipolytica. An analysis of trafficking of peroxisomal proteins in vivo suggests the existence of a multistep peroxisome assembly pathway in Y. lipolytica. This pathway operates by conversion of peroxisomal subforms in the direction P1, P2-->P3-->P4-->P5-->P6 and involves the import of various peroxisomal proteins into distinct vesicular intermediates. We have also reconstituted in vitro the fusion of the earliest intermediates in the pathway, small peroxisomal vesicles P1 and P2. Their fusion leads to the formation of a larger and more dense peroxisomal vesicle, P3. Fusion of P1 and P2 in vitro requires cytosol and ATP hydrolysis and is inhibited by antibodies to two membrane-associated ATPases of the AAA family, Pex1p and Pex6p. We provide evidence that the fusion in vitro of P1 and P2 peroxisomes reconstructs an actual early step in the peroxisome assembly pathway operating in vivo in Y. lipolytica.     

Peroxisomal Targeting of PTS2 Pre-Import Complexes in the Yeast Saccharomyces cerevisiae

Posttranslational matrix protein import into peroxisomes uses either one of the two peroxisomal targeting signals (PTS), PTS1 and PTS2. Unlike the PTS1 receptor Pex5p, the PTS2 receptor Pex7p is necessary but not sufficient to target cargo proteins into the peroxisomal matrix and requires coreceptors. Saccharomyces cerevisiae possesses two coreceptors, Pex18p and Pex21p, with a redundant but not a clearly defined function. To gain further insight into the early events of this import pathway, PTS2 pre-import complexes of S. cerevisiae were isolated and characterized by determination of size and protein composition in wild-type and different mutant strains. Mass spectrometric analysis of the cytosolic PTS2 pre-import complex indicates that Fox3p is the only abundant PTS2 protein under oleate growth conditions. Our data strongly suggest that the formation of the ternary cytosolic PTS2 pre-import complex occurs hierarchically. First, Pex7p recognizes cargo proteins through its PTS2 in the cytosol. In a second step, the coreceptor binds to this complex, and finally, this ternary 150 kDa pre-import complex docks at the peroxisomal membrane, where both the PTS1 and the PTS2 import pathways converge. Gel filtration analysis of membrane-bound subcomplexes suggests that Pex13p provides the initial binding partner at the peroxisomal membrane, whereas Pex14p assembles with Pex18p in high-molecular-weight complexes after or during dissociation of the PTS2 receptor.     

Saccharomyces cerevisiae pex3p and pex19p are required for proper localization and stability of peroxisomal membrane proteins

The mechanisms by which peroxisomal membrane proteins (PMPs) are targeted to and inserted into membranes are unknown, as are the required components. We show that among a collection of 16 Saccharomyces cerevisiae peroxisome biogenesis (pex) mutants, two mutants, pex3Delta and pex19Delta, completely lack detectable peroxisomal membrane structures and mislocalize their PMPs to the cytosol where they are rapidly degraded. The other pexDelta mutants contain membrane structures that are properly inherited during vegetative growth and that house multiple PMPs. Even Pex15p requires Pex3p and Pex19p for localization to peroxisomal membranes. This PMP was previously hypothesized to travel via the endoplasmic reticulum (ER) to peroxisomes. We provide evidence that ER-accumulated Pex15p is not a sorting intermediate on its way to peroxisomes. Our results show that Pex3p and Pex19p are required for the proper localization of all PMPs tested, including Pex15p, whereas the other Pex proteins might only be required for targeting/import of matrix proteins.