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.     

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.     

Formation of peroxisomes from peroxisomal ghosts in a peroxisome-deficient mammalian cell mutant upon complementation by protein microinjection

Most mammalian cell strains genetically deficient in peroxisome biogenesis have abnormal membrane structures called ghosts, containing integral peroxisomal membrane protein, PMP70, but lacking the peroxisomal matrix proteins. Upon genetic complementation, these mutants regain the ability of peroxisome biogenesis. It is postulated that, in this process, the ghosts act as the precursors of peroxisomes, but there has been no evidence to support this. In the present study, we investigated this issue by protein microinjection to a mutant Chinese hamster ovary cell line defective of PEX5, encoding a peroxisome-targeting signal receptor. When recombinant Pex5p and green fluorescent protein (GFP) carrying a peroxisome-targeting signal were co-injected into the mutant cells, the GFP fluorescence gathered over time to particulate structures where PMP70 was co-localized. This process was dependent on both Pex5p and the targeting signal, and, most importantly, occurred even in the presence of cycloheximide, a protein synthesis inhibitor. These findings suggest that the ghosts act as acceptors of matrix proteins in the peroxisome recovery process at least in the PEX5 mutant, and support the view that peroxisomes can grow by incorporating newly synthesized matrix proteins.     

The Hansenula polymorpha PEX14 gene encodes a novel peroxisomal membrane protein essential for peroxisome biogenesis.

We have cloned the Hansenula polymorpha PEX14 gene by functional complementation of the chemically induced pex14-1 mutant, which lacked normal peroxisomes. The sequence of the PEX14 gene predicts a novel protein product (Pex14p) of 39 kDa which showed no similarity to any known protein and lacked either of the two known peroxisomal targeting signals. Biochemical and electron microscopical analysis indicated that Pex14p is a component of the peroxisomal membrane. The synthesis of Pex14p is induced by peroxisome-inducing growth conditions. In cells of both pex14-1 and a PEX14 disruption mutant, peroxisomal membrane remnants were evident; these contained the H.polymorpha peroxisomal membrane protein Pex3p together with a small amount of the major peroxisomal matrix proteins alcohol oxidase, catalase and dihydroxyacetone synthase, the bulk of which resided in the cytosol. Unexpectedly, overproduction of Pex14p in wild-type H. polymorpha cells resulted in a peroxisome-deficient phenotype typified by the presence of numerous small vesicles which lacked matrix proteins; these were localized in the cytosol. Apparently, the stoichiometry of Pex14p relative to one or more other components of the peroxisome biogenesis machinery appears to be critical for protein 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.     

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.     

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.     

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.     

The membrane biogenesis peroxin Pex16p. Topogenesis and functional roles in peroxisomal membrane assembly

Previously we isolated human PEX16 encoding 336-amino acid-long peroxin Pex16p and showed that its dysfunction was responsible for Zellweger syndrome of complementation group D (group 9). Here we have determined the membrane topology of Pex16p by differential permeabilization method: both N- and C-terminal parts are exposed to the cytosol. In the search for Pex16p topogenic sequence, basic amino acids clustered sequence, RKELRKKLPVSLSQQK, at positions 66-81 and the first transmembrane segment locating far downstream, nearly by 40 amino acids, of this basic region were defined to be essential for integration into peroxisome membranes. Localization to peroxisomes of membrane proteins such as Pex14p, Pex13p, and PMP70 was interfered with in CHO-K1 cells by a higher level expression of the pex16 patient-derived dysfunctional but topogenically active Pex16pR176ter comprising resides 1-176 or of the C-terminal cytoplasmic part starting from residues at 244 to the C terminus. Furthermore, Pex16p C-terminal cytoplasmic part severely abrogated peroxisome restoration in pex mutants such as matrix protein import-defective pex12 and membrane assembly impaired pex3 by respective PEX12 and PEX3 expression, whereas the N-terminal cytosolic region did not affect restoration. These results imply that Pex16p functions in peroxisome membrane assembly, more likely upstream of Pex3p.     

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.     

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.     

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.     

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.     

Conserved function of pex11p and the novel pex25p and pex27p in peroxisome biogenesis

We describe the isolation and characterization of a homologous pair of proteins, Pex25p (YPL112c) and Pex27p (YOR193w), whose C-termini are similar to the entire Pex11p. All three proteins localize to the peroxisomal membrane and are likely to form homo-oligomers. Deletion of any of the three genes resulted in enlarged peroxisomes as revealed by fluorescence and electron microscopy. The partial growth defect on fatty acids of a pex25Δ mutant was not exacerbated by the additional deletion of PEX27; however, when PEX11 was deleted on top of that, growth was abolished on all fatty acids. Moreover, a severe peroxisomal protein import defect was observed in the pex11Δpex25Δpex27Δ triple mutant strain. This import defect was also observed when cells were grown on ethanol-containing medium, where peroxisomes are not required, suggesting that the function of the proteins in peroxisome biogenesis exceeds their role in proliferation. When Pex25p was overexpressed in the triple mutant strain, growth on oleic acid was completely restored and a massive proliferation of laminar membranes and peroxisomes was observed. Our data demonstrate that Pex11p, Pex25p, and Pex27p build a family of proteins whose members are required for peroxisome biogenesis and play a role in the regulation of peroxisome size and number.     

Yarrowia lipolytica cells mutant for the PEX24 gene encoding a peroxisomal membrane peroxin mislocalize peroxisomal proteins and accumulate membrane structures containing both peroxisomal matrix and membrane proteins

Peroxins are proteins required for peroxisome assembly and are encoded by the PEX genes. Functional complementation of the oleic acid-nonutilizing strain mut1-1 of the yeast Yarrowia lipolytica has identified the novel gene, PEX24. PEX24 encodes Pex24p, a protein of 550 amino acids (61,100 Da). Pex24p is an integral membrane protein of peroxisomes that exhibits high sequence homology to two hypothetical proteins encoded by the open reading frames YHR150W and YDR479C of the Saccharomyces cerevisiae genome. Pex24p is detectable in wild-type cells grown in glucose-containing medium, and its levels are significantly increased by incubation of cells in oleic acid-containing medium, the metabolism of which requires intact peroxisomes. pex24 mutants are compromised in the targeting of both matrix and membrane proteins to peroxisomes. Although pex24 mutants fail to assemble functional peroxisomes, they do harbor membrane structures that contain subsets of peroxisomal proteins.     

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.     

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.     

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.     

Critical role of the peroxisomal protein PEX16 in white adipocyte development and lipid homeostasis

The importance of peroxisomes for adipocyte function is poorly understood. Herein, we provide insights into the critical role of peroxin 16 (PEX16)-mediated peroxisome biogenesis in adipocyte development and lipid metabolism. Pex16 is highly expressed in adipose tissues and upregulated during adipogenesis of murine and human cells. We demonstrate that Pex16 is a target gene of the adipogenesis “master-regulator” PPARγ. Stable silencing of Pex16 in 3T3-L1 cells strongly reduced the number of peroxisomes while mitochondrial number was unaffected. Concomitantly, peroxisomal fatty acid (FA) oxidation was reduced, thereby causing accumulation of long- and very long-chain (polyunsaturated) FAs and reduction of odd-chain FAs. Further, Pex16-silencing decreased cellular oxygen consumption and increased FA release. Additionally, silencing of Pex16 impaired adipocyte differentiation, lipogenic and adipogenic marker gene expression, and cellular triglyceride stores. Addition of PPARγ agonist rosiglitazone and peroxisome-related lipid species to Pex16-silenced 3T3-L1 cells rescued adipogenesis. These data provide evidence that PEX16 is required for peroxisome biogenesis and highlights the relevance of peroxisomes for adipogenesis and adipocyte lipid metabolism.     

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.