Regulation mechanism of ERM (ezrin/radixin/moesin) protein/plasma membrane association: possible involvement of phosphatidylinositol turnover and Rho-dependent signaling pathway

Candida boidinii Pmp47, an integral peroxisomal membrane protein, belongs to a family of mitochondrial solute transporters (e.g., ATP/ADP exchanger), and is the only known peroxisomal member of this family. However, its physiological and biochemical functions have been unrevealed because of the difficulties in the molecular genetics of C. boidinii. In this study, we first isolated the PMP47 gene, which was the single gene encoding for Pmp47 in a gene-engineerable strain S2 of C. boidinii. Sequence analysis revealed that it was very similar to PMP47A and PMP47B genes from a polyploidal C. Boidinii strain (ATCC32195). Next, the PMP47 gene was disrupted and the disruption strain (pmp47delta) was analyzed. Depletion of PMP47 from strain S2 resulted in a retarded growth on oleate and a complete loss of growth on methanol. Both growth substrates require peroxisomal metabolism. EM observations revealed the presence of peroxisomes in methanol- and oleate-induced cells of pmp47delta, but in reduced numbers, and the presence of material of high electron density in the cytoplasm in both cases. Methanol-induced cells of pmp47delta were investigated in detail. The activity of one of the methanol-induced peroxisome matrix enzymes, dihydroxyacetone synthase (DHAS), was not detected in pmp47delta. Further biochemical and immunocytochemical experiments revealed that the DHAS protein aggregated in the cytoplasm as an inclusion body, while two other peroxisome matrix enzymes, alcohol oxidase (AOD) and catalase, were active and found in peroxisomes. Two peroxisome-deficient mutants, strains M6 and M13 (described in previous studies), retained DHAS activity although it was mislocalized to the cytoplasm and the nucleus. We disrupted PMP47 in these peroxisome- deficient mutants. In both strains, M6-pmp47delta and M13-pmp47delta, DHAS was enzymatically active and was located in the cytoplasm and the nucleus. We suggest that an unknown small molecule, which PMP47 transports, is necessary for the folding or the translocation machinery of DHAS within peroxisomes. Pmp47 does not catalyze folding directly because active DHAS is observed in the M6-pmp47delta and M13-pmp47delta strains. Since both AOD and DHAS have the PTS1 motif sequences at their carboxyl terminal, our results first show that depletion of Pmp47 could dissect the peroxisomal import pathway (PTS1 pathway) of these proteins.     

Pmp27 promotes peroxisomal proliferation

Peroxisomes perform many essential functions in eukaryotic cells. The weight of evidence indicates that these organelles divide by budding from preexisting peroxisomes. This process is not understood at the molecular level. Peroxisomal proliferation can be induced in Saccharomyces cerevisiae by oleate. This growth substrate is metabolized by peroxisomal enzymes. We have identified a protein, Pmp27, that promotes peroxisomal proliferation. This protein, previously termed Pmp24, was purified from peroxisomal membranes, and the corresponding gene, PMP27, was isolated and sequenced. Pmp27 shares sequence similarity with the Pmp30 family in Candida boidinii. Pmp27 is a hydrophobic peroxisomal membrane protein but it can be extracted by high pH, suggesting that it does not fully span the bilayer. Its expression is regulated by oleate. The function of Pmp27 was probed by observing the phenotype of strains in which the protein was eliminated by gene disruption or overproduced by expression from a multicopy plasmid. The strain containing the disruption (3B) was able to grow on all carbon sources tested, including oleate, although growth on oleate, glycerol, and acetate was slower than wild type. Strain 3B contained peroxisomes with all of the enzymes of beta-oxidation. However, in addition to the presence of a few modestly sized peroxisomes seen in a typical thin section of a cell growing on oleate-containing medium, cells of strain 3B also contained one or two very large peroxisomes. In contrast, cells in a strain in which Pmp27 was overexpressed contained an increased number of normal-sized peroxisomes. We suggest that Pmp27 promotes peroxisomal proliferation by participating in peroxisomal elongation or fission.     

Peroxisomal membrane protein Pmp47 is essential in the metabolism of middle-chain fatty acid in yeast peroxisomes and Is associated with peroxisome proliferation

Pmp47 of the methylotrophic yeast Candida boidinii belongs to a mitochondrial family of solute transporters and is localized in peroxisomal membranes. Its human homolog, Pmp34, is also known. In this study, we characterized the role of Pmp47 in fatty acid metabolism and peroxisome proliferation using the PMP47-deleted strain of C. boidinii (strain pmp47Delta). The wild-type strain grew well on a middle-chain fatty acid, laureate, as the single carbon source, and mild peroxisome proliferation was observed during its growth. The pmp47Delta strain could not grow on laureate but could grow on long-chain fatty acids including palmitate, myristate, and oleate. The levels of laureate oxidation activity in intact cells and in semi-permeabilized cells of strain pmp47Delta were lower than the respective level in the wild-type strain, although the level of laureate oxidation activity in the cell lysate and the level of lauroyl-CoA oxidation in semi-permeabilized cells of strain pmp47Delta were indistinguishable from the respective level in the wild-type strain. When lauroyl-CoA was provided in the cytosol of strain pmp47Delta through expression of Saccharomyces cerevisiae Faa2p (lauroyl-CoA synthetase) in which its peroxisome targeting signal was deleted, the growth of strain pmp47Delta on laureate was recovered to the level of growth of the wild-type strain. Laureate is converted to its CoA form in peroxisomes by the action of lauroyl-CoA synthetase. These results suggested that Pmp47 is involved in the transport of a small molecule (possibly ATP) required in the conversion of laureate to its CoA form in peroxisomes and that the absence of Pmp47 causes impairment of laureate metabolism, which results in the inability of pmp47Delta cells to grow on laureate. In addition, Pmp47 may be involved in peroxisome proliferation, because the pmp47Delta strain contained a reduced number of peroxisomes, as judged from the fluorescence analysis of cells expressing green fluorescent protein tagged with the peroxisome targeting signal 1 (GFP-AKL).     

Absence of the peroxiredoxin Pmp20 causes peroxisomal protein leakage and necrotic cell death

We analyzed the role of the peroxisomal peroxiredoxin Pmp20 of the yeast Hansenula polymorpha. Cells of a PMP20 disruption strain (pmp20) grew normally on substrates that are not metabolized by peroxisomal enzymes, but showed a severe growth defect on methanol, the metabolism of which involves a hydrogen peroxide producing peroxisomal oxidase. This growth defect was paralleled by leakage of peroxisomal matrix proteins into the cytosol. Methanol-induced pmp20 cells accumulated enhanced levels of reactive oxygen species and lipid peroxidation products. Moreover, the fatty acid composition of methanol-induced pmp20 cells differed relative to WT controls, suggesting an effect on fatty acid homeostasis. Plating assays and FACS-based analysis of cell death markers revealed that pmp20 cells show loss of clonogenic efficiency and membrane integrity, when cultured on methanol. We conclude that the absence of the peroxisomal peroxiredoxin leads to loss of peroxisome membrane integrity and necrotic cell death.     

The Candida boidinii peroxisomal membrane protein Pmp30 has a role in peroxisomal proliferation and is functionally homologous to Pmp27 from Saccharomyces cerevisiae.

The mechanism of peroxisome proliferation is poorly understood. Candida boidinii is a methylotrophic yeast that undergoes rapid and massive peroxisome proliferation and serves as a good model system for this process. Pmp30A and Pmp30B (formerly designated Pmp31 and Pmp32, respectively) are two closely related proteins in a polyploid strain of this yeast that are strongly induced by diverse peroxisome proliferators such as methanol, oleate, and D-alanine. The function of these proteins is not understood. To study this issue, we used a recently described haploid strain (S2) of C. boidinii that can be manipulated genetically. We now report that strain S2 contains a single PMP30 gene very similar in sequence (greater than 93% identity at the DNA level) to PMP30A and PMP30B. When PMP30 was disrupted, cell growth on methanol was greatly inhibited, and cells grown in both methanol and oleate had fewer, larger, and more spherical peroxisomes than wild-type cells. A similar phenotype was recently described for Saccharomyces cerevisiae cultured on oleate in which PMP27, which encodes a protein of related sequence that is important for peroxisome proliferation, was disrupted. To determine whether Pmp27 is a functional homolog of Pmp30, gentle complementation was performed. PMP30A was expressed in the PMP27 disruptant of S. cerevisiae, and PMP27 was expressed in the PMP30 disruptant of C. boidinii S2. Complementation, in terms of both cell growth and organelle size, shape, and number, was successful in both directions, although reversion to a wild-type phenotype was only partial for the PMP30 disruptant. We conclude that these proteins are functional homologs and that both Pmp30 and Pmp27 have a direct role in proliferation and organelle size rather than a role in a specific peroxisomal metabolic pathway of substrate utilization.     

Antioxidant system within yeast peroxisome. Biochemical and physiological characterization of CbPmp20 in the methylotrophic yeast Candida boidinii

Candida boidinii Pmp20 (CbPmp20), a protein associated with the inner side of peroxisomal membrane, belongs to a recently identified protein family of antioxidant enzymes, the peroxiredoxins, which contain one cysteine residue. Pmp20 homologs containing the putative peroxisome targeting signal type 1 have also been identified in mammals and lower eukaryotes. However, the physiological function of these Pmp20 family proteins has been unclear. In this study, we investigated the biochemical and physiological functions of recombinant CbPmp20 protein in methanol-induced peroxisomes of C. boidinii using the PMP20-deleted strain of C. boidinii (pmp20Delta strain). The His(6)-tagged CbPmp20 fusion protein was found to have glutathione peroxidase activity in vitro toward alkyl hydroperoxides and H(2)O(2). Catalytic activity and dimerization of His(6)-CbPmp20 depended on the only cysteine residue corresponding to Cys(53). The pmp20Delta strain was found to have lost growth ability on methanol as a carbon and energy source. The pmp20Delta growth defect was rescued by CbPmp20, but neither CbPmp20 lacking the peroxisome targeting signal type 1 sequence nor CbPmp20 haboring the C53S mutation retrieved the growth defect. Interestingly, the pmp20Delta strain had a more severe growth defect than the cta1Delta strain, which lacks catalase, another antioxidant enzyme within the peroxisome. During incubation of these strains in methanol medium, the cta1Delta strain accumulated H(2)O(2), whereas the pmp20Delta strain did not. Therefore, it is speculated to be the main function of CbPmp20 is to decompose reactive oxygen species generated at peroxisomal membrane surface, e.g. lipid hydroperoxides, rather than to decompose H(2)O(2). In addition, we detected a physiological level of reduced glutathione in peroxisomal fraction of C. boidinii. These results may indicate a physiological role for CbPmp20 as an antioxidant enzyme within peroxisomes rich in reactive oxygen species.     

Identification of Pex13p a peroxisomal membrane receptor for the PTS1 recognition factor

We have identified an S. cerevisiae integral peroxisomal membrane protein of M of 42,705 (Pex13p) that is a component of the peroxisomal protein import apparatus. Pex13p's most striking feature is an src homology 3 (SH3) domain that interacts directly with yeast Pex5p (former Pas10p), the recognition factor for the COOH-terminal tripeptide signal sequence (PTS1), but not with Pex7p (former Pas7p), the recognition factor for the NH2-terminal nonapeptide signal (PTS2) of peroxisomal matrix proteins. Hence, Pex13p serves as peroxisomal membrane receptor for at least one of the two peroxisomal signal recognition factors. Cells deficient in Pex13p are unable to import peroxisomal matrix proteins containing PTS1 and, surprisingly, also those containing PTS2. Pex13p deficient cells retain membranes containing the peroxisomal membrane protein Pex11p (former Pmp27p), consistent with the existence of independent pathways for the integration of peroxisomal membrane proteins and for the translocation of peroxisomal matrix proteins.     

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.     

Sorting of peroxisomal membrane protein PMP47 from Candida boidinii into peroxisomal membranes of Saccharomyces cerevisiae

A gene encoding PMP47, a peroxisomal integral membrane protein of the methylotrophic yeast Candida boidinii, was isolated from a genomic library. DNA sequencing of PMP47 revealed an open reading frame of 1269 base pairs capable of encoding a protein of 46,873 Da. At least two membrane-spanning regions in the protein are predicted from the sequence. Since the 3 amino acids at the carboxyl terminus are -AKE, PMP47 lacks a typical peroxisomal sorting signal. No significant similarities in primary structure between PMP47 and known proteins were observed, including PMP70, a rat peroxisomal membrane protein whose sequence has recently been reported (Kamijo, K., Taketani, S., Yokota, S., Osumi, T., and Hashimoto, T. (1990). J. Biol. Chem. 265, 4534-4540). In order to study the import and assembly of PMP47 into peroxisomes by genetic approaches, the gene was expressed in the yeast Saccharomyces cerevisiae. When PMP47 was expressed in cells grown on oleic acid to induce peroxisomes, the protein was observed exclusively in peroxisomes as determined by marker enzyme analysis of organelle fractions. Most of the PMP47 co-purified with the endogenous peroxisomal membrane proteins on isopycnic sucrose gradients. Either in the native host or when expressed in S. cerevisiae, PMP47 was not extractable from peroxisomal membranes by sodium carbonate at pH 11, indicating an integral membrane association. These results indicate that PMP47 is competent for sorting to and assembling into peroxisomal membranes in S. cerevisiae.     

Characterization of the 70-kDa peroxisomal membrane protein, an ATP binding cassette transporter

The 70-kDa peroxisomal membrane protein (PMP70) is one of the major components of rat liver peroxisomal membranes and belongs to a superfamily of proteins known as ATP binding cassette transporters. PMP70 is markedly induced by administration of hypolipidemic agents in parallel with peroxisome proliferation and induction of peroxisomal fatty acid beta-oxidation enzymes. To characterize the role of PMP70 in biogenesis and function of peroxisomes, we transfected the cDNA of rat PMP70 into Chinese hamster ovary cells and established cell lines stably expressing PMP70. The content of PMP70 in the transfectants increased about 5-fold when compared with the control cells. A subcellular fractionation study showed that overexpressed PMP70 was enriched in peroxisomes. This peroxisomal localization was confirmed by immunofluorescence and immunoelectron microscopy. The number of immuno-gold particles corresponding to PMP70 on peroxisomes increased markedly in the transfectants, but the size and the number of peroxisomes were essentially the same in both the transfectants and the control cells. beta-Oxidation of palmitic acid increased about 2-3-fold in the transfectants, whereas the oxidation of lignoceric acid decreased about 30-40%. When intact peroxisomes prepared from both the cell lines were incubated with palmitoyl-CoA, oxidation was stimulated with ATP, but the degree of the stimulation was higher in the transfectants than in the control cells. Furthermore, we established three Chinese hamster ovary cell lines stably expressing mutant PMP70. In these cells, beta-oxidation of palmitic acid decreased markedly. These results suggest that PMP70 is involved in metabolic transport of long chain acyl-CoA across peroxisomal membranes and that increase of PMP70 is not associated with proliferation of peroxisomes.     

Redox-sensitive homodimerization of Pex11p: a proposed mechanism to regulate peroxisomal division

Pex11p (formerly Pmp27) has been implicated in peroxisomal proliferation (Erdmann, R., and G. Blobel. 1995. J. Cell Biol. 128; 509- 523; Marshall, P.A., Y.I. Krimkevich, R.H. Lark, J.M. Dyer, M. Veenhuis, and J.M. Goodman, 1995. J. Cell Biol. 129; 345-355). In its absence, peroxisomes in Saccharomyces cerevisiae fail to proliferate in response to oleic acid; instead, one or two large peroxisomes are formed. Conversely, overproduction of Pex11p causes an increase in peroxisomal number. In this report, we confirm the function of Pex11p in organelle proliferation by demonstrating that this protein can cause fragmentation in vivo of large peroxisomes into smaller organelles. Pex11p is on the inner surface of the peroxisomal membrane. It can form homodimers, and this species is more abundant in mature peroxisomes than in proliferating organelles. Removing one of the three cysteines in the protein inhibits homodimerization. This cysteine 3-->alanine mutation leads to an increase in number and a decrease in peroxisomal density, compared with the wild-type protein, in response to oleic acid. We propose that the active species is the "monomeric" form, and that the increasing oxidative metabolism within maturing peroxisomes causes dimer formation and inhibition of further organelle division.     

Targeting of the 22 kDa integral peroxisomal membrane protein

Investigating targeting of the 22 kDa peroxisomal membrane protein (Pmp22p) to the peroxisomal membrane we have confined the targeting signal to amino acid residues 16-37 located in the N-terminal cytoplasmic tail. Comparison of Pmp22p orthologous sequences revealed a conserved motif Y3xL3xP3x(KQN) which might represent the core of this targeting signal not found so far in other Pmps. Fusion of the Pmp22p N-terminal tail to the C-terminal portion of Pmp22p which per se is not targeted to peroxisomes, conveys peroxisomal targeting. These data suggest that Pmp22p is targeted to peroxisomes by a new membrane targeting signal which is necessary and sufficient to target a polypeptide containing two transmembrane spans to peroxisomes.     

Association of glyoxylate and beta-oxidation enzymes with peroxisomes of Saccharomyces cerevisiae.

Although peroxisomes are difficult to identify in Saccharomyces cerevisiae under ordinary growth conditions, they proliferate when cells are cultured on oleic acid. We used this finding to study the protein composition of these organelles in detail. Peroxisomes from oleic acid-grown cells were purified on a discontinuous sucrose gradient; they migrated to the 46 to 50% (wt/wt) sucrose interface. The peroxisomal fraction was identified morphologically and by the presence of all of the enzymes of the peroxisomal beta-oxidation pathway. These organelles also contained a significant but minor fraction of two enzymes of the glyoxylate pathway, malate synthase and malate dehydrogenase-2. The localization of malate synthase in peroxisomes was confirmed by immunoelectron microscopy. It is postulated that glyoxylate pathway enzymes are readily and preferentially released from peroxisomes upon cell lysis, accounting for their incomplete recovery from isolated organelles. Small uninduced peroxisomes from glycerol-grown cultures were detected on sucrose gradients by marker enzymes. Under these conditions, catalase, acyl-coenzyme A oxidase, and malate synthase cofractionated at equilibrium close to the mitochondrial peak, indicating smaller, less dense organelles than those from cells grown on oleic acid. Peroxisomal membranes from oleate cultures were purified by buoyant density centrifugation. Three abundant proteins of 24, 31, and 32 kilodaltons were observed.     

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.     

Discrete targeting signals direct Pmp47 to oleate-induced peroxisomes in Saccharomyces cerevisiae

Pmp47 is a peroxisomal membrane protein consisting of six transmembrane domains (TMDs). We previously showed that the second matrix loop containing a basic cluster of amino acids is important for peroxisomal targeting, and similar basic targeting motifs have been found in other peroxisomal membrane proteins. However, this basic cluster by itself targets to peroxisomes very poorly. We have developed a sensitive quantitative localization assay based on the targeting of Pmp47-GFP fusion proteins to identify the important elements of the basic cluster and to search for other targeting information on Pmp47. Our data suggest that side-chain structure and position as well as charge are important for targeting by the basic cluster. Analysis of other regions of Pmp47 indicates that all TMDs except TMD2 can be eliminated or substituted without significant loss of targeting. TMD2 plus an adjacent cytoplasmic-oriented sequence is crucial for targeting. Cytoplasmic-oriented sequences from two other peroxisomal membrane proteins, ScPex15p and ScPmp22, could partially substitute for the analogous sequence in Pmp47. Targeting with high fidelity to oleate-induced peroxisomes required the following elements: the cytoplasmic-oriented sequence and TMD2, a short matrix loop containing a basic cluster, and a membrane-anchoring TMD.     

Multiple targeting modules on peroxisomal proteins are not redundant: discrete functions of targeting signals within Pmp47 and Pex8p

Several peroxisomal proteins have two nonoverlapping targeting signals. These signals have been termed “redundant” because targeting can still occur with only one signal. We now report that separate targeting motifs within both Pmp47 and Pex8 provide complementary function. Pmp47 is an ATP translocator that contains six transmembrane domains (TMDs). We had previously shown that the TMD2 region (termed TMD2R, consisting of TMD2 and a short adjacent segment of cytosolic loop) was required for targeting to proliferated peroxisomes in Saccharomyces cerevisiae. We now report that the analogous TMD4R, which cannot target to proliferated peroxisomes, targets at least as well, or much better (depending on strain and growth conditions) in cells containing only basal (i.e., nonproliferated) peroxisomes. These data suggest differences in the targeting pathway among peroxisome populations. Pex8p, a peripheral protein facing the matrix, contains a typical carboxy terminal targeting sequence (PTS1) that has been shown to be nonessential for targeting, indicating the existence of a second targeting domain (not yet defined in S. cerevisiae); thus, its function was unknown. We show that targeting to basal peroxisomes, but not to proliferated peroxisomes, is more efficient with the PTS1 than without it. Our results indicate that multiple targeting signals within peroxisomal proteins extend coverage among heterogeneous populations of peroxisomes and increase efficiency of targeting in some metabolic states.     

Lipid composition of peroxisomes from the yeast Pichia pastoris grown on different carbon sources

Highly purified peroxisomes from the yeast Pichia pastoris grown on methanol or oleic acid, respectively, were used to characterize the lipid composition of this organelle. For this purpose, an isolation procedure had to be adapted which yielded highly purified P. pastoris peroxisomes. When peroxisome proliferation was induced by growth on methanol, alcohol oxidase was the predominant peroxisomal protein. Cultivation of P. pastoris on oleic acid led to induction of a family of peroxisomal enzymes catalyzing fatty acid beta-oxidation, whose most prominent members were identified by mass spectrometry. On either carbon source, phosphatidylcholine and phosphatidylethanolamine were the major peroxisomal phospholipids, and cardiolipin was present in peroxisomal membranes at a substantial amount, indicating that this phospholipid is a true peroxisomal component. Ergosterol was the most abundant sterol of P. pastoris peroxisomal membranes irrespective of the culture conditions. The fatty acid composition of whole cells and peroxisomes was highly affected by cultivation of P. pastoris on oleic acid. Under these conditions, oleic acid became the predominant fatty acid in phospholipids from total cell and peroxisomal extracts. Thus, oleic acid was not only utilized as an appropriate carbon source but also as a building block for complex membrane lipids. In summary, our data provide first insight into biochemical properties of P. pastoris peroxisomal membranes, which may become important for the biotechnological use of this yeast.     

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.     

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.