Characterization of a novel component of the peroxisomal protein import apparatus using fluorescent peroxisomal proteins.

Fluorescent peroxisomal probes were developed by fusing green fluorescent protein (GFP) to the matrix peroxisomal targeting signals PTS1 and PTS2, as well as to an integral peroxisomal membrane protein (IPMP). These proteins were used to identify and characterize novel peroxisome assembly (pas) mutants in the yeast Pichia pastoris. Mutant cells lacking the PAS10 gene mislocalized both PTS1-GFP and PTS2-GFP to the cytoplasm but did incorporate IPMP-GFP into peroxisome membranes. Similar distributions were observed for endogenous peroxisomal matrix and membrane proteins. While peroxisomes from translocation-competent pas mutants sediment in sucrose gradients at the density of normal peroxisomes, >98% of peroxisomes from pas10 cells migrated to a much lower density and had an extremely low ratio of matrix:membrane protein. These data indicate that Pas10p plays an important role in protein translocation across the peroxisome membrane. Consistent with this hypothesis, we find that Pas10p is an integral protein of the peroxisome membrane. In addition, Pas10p contains a cytoplasmically-oriented C3HC4 zinc binding domain that is essential for its biological activity.     

Toward a definition of the complete proteome of plant peroxisomes: Where experimental proteomics must be complemented by bioinformatics

In the past few years, proteome analysis of Arabidopsis peroxisomes has been established by the complementary efforts of four research groups and has emerged as the major unbiased approach to identify new peroxisomal proteins on a large scale. Collectively, more than 100 new candidate proteins from plant peroxisomes have been identified, including long-awaited low-abundance proteins. More than 50 proteins have been validated as peroxisome targeted, nearly doubling the number of established plant peroxisomal proteins. Sequence homologies of the new proteins predict unexpected enzyme activities, novel metabolic pathways and unknown non-metabolic peroxisome functions. Despite this remarkable success, proteome analyses of plant peroxisomes remain highly material intensive and require major preparative efforts. Characterization of the membrane proteome or post-translational protein modifications poses major technical challenges. New strategies, including quantitative mass spectrometry methods, need to be applied to allow further identifications of plant peroxisomal proteins, such as of stress-inducible proteins. In the long process of defining the complete proteome of plant peroxisomes, the prediction of peroxisome-targeted proteins from plant genome sequences emerges as an essential complementary approach to identify additional peroxisomal proteins that are, for instance, specific to peroxisome variants from minor tissues and organs or to abiotically stressed model and crop plants.     

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.     

Insights into the membrane proteome of rat liver peroxisomes: microsomal glutathione-S-transferase is shared by both subcellular compartments

Peroxisomes are ubiquitous "multipurpose" organelles of eukaryotic cells. Their matrix enzymes catalyze mainly catabolic and anabolic reactions of lipid metabolism, thus contributing to the regulation of lipid homeostasis. Since most metabolites must be actively transported across the peroxisomal membrane and since individual proteins and protein complexes play functional roles in such transport processes, we analyzed the peroxisomal membrane proteome. Benzyldimethyl-n-hexadecylammoniumchloride (16-BAC)/SDS-2-D-PAGE and mass spectrometry were used to characterize the proteomes of highly purified "light" and "heavy" peroxisomes of rat liver obtained by density gradient centrifugation. In both populations, the major integral membrane proteins could be detected in high concentrations, verifying 16-BAC/SDS-2-D-PAGE as a suitable tool for the preparation of membrane proteomes destined for mass spectrometric analysis. Both reliable and reproducible detection of a distinct set of microsomal (ER) membrane proteins, including microsomal glutathione-S-transferase (mGST), in light and heavy peroxisomal fractions was also possible. Compared with the abundance of most microsomal membrane proteins, we found mGST to be specifically enriched in peroxisomal membrane fractions. Furthermore, C terminus epitope-tagged mGST versions were localized at least in part to peroxisomes in different mammalian cell lines. Taken together, these data suggest that the peroxisomal GST is not a mere ER-contaminant, but a bona fide protein comprising the membrane proteome of both intracellular compartments. In addition, we could detect several mitochondrial proteins in light peroxisome fractions. This finding may likely indicate a physical association of light peroxisomes with mitochondria, since the organelles could be partly separated by mechanical stress. Whether this association is of functional importance awaits further investigation.     

A novel method to determine the topology of peroxisomal membrane proteins in vivo using the tobacco etch virus protease.

Most proteins essential for the biogenesis of peroxisomes (peroxins) that are identified to date are associated with or are integral components of the peroxisomal membrane. A prerequisite in elucidating their function is to determine their topology in the membrane. We have developed a novel tool to analyze the topology of peroxisomal membrane proteins in the yeast Hansenula polymorpha in vivo using the 27-kDa NIa protease subunit from the tobacco etch virus (TEVp). TEVp specifically cleaves peptides containing the consensus sequence, EXXYXQ downward arrowS (tev). We show that cytosolic TEVp and peroxisomal TEVp.SKL are selectively active on soluble cytosolic and peroxisomal tev-containing proteins in vivo, respectively, without affecting the viability of the yeast cells. The tev sequence was introduced in between the primary sequence of the peroxisomal membrane proteins Pex3p or Pex10p and the reporter protein enhanced green fluorescent protein (eGFP). Co-synthesis of these functional tev-GFP tagged proteins with either cytosolic TEVp or peroxisomal TEVp.SKL revealed that the C termini of Pex3p and Pex10p are exposed to the cytosol. Additional applications of the TEV protease to study peroxisome biogenesis are discussed.     

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.     

Pex19p contributes to peroxisome inheritance in the association of peroxisomes to Myo2p

During budding of yeast cells peroxisomes are distributed over mother cell and bud, a process that involves the myosin motor protein Myo2p and the peroxisomal membrane protein Inp2p. Here, we show that Pex19p, a peroxin implicated in targeting and complex formation of peroxisomal membrane proteins, also plays a role in peroxisome partitioning. Binding studies revealed that Pex19p interacts with the cargo-binding domain of Myo2p. We identified mutations in Myo2p that specifically reduced binding to Pex19p, but not to Inp2p. The interaction between Myo2p and Pex19p was also reduced by a mutation that blocked Pex19p farnesylation. Microscopy revealed that the Pex19p-Myo2p interaction is important for peroxisome inheritance, because mutations that affect this interaction hamper peroxisome inheritance in vivo. Together these data suggest that both Inp2p and Pex19p are required for proper association of peroxisomes to Myo2p.     

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.     

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.     

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.     

Expression of the Salmonella Spp. Virulence Factor SifA in Yeast Alters Rho1 Activity on Peroxisomes

The Salmonella typhimurium effector protein SifA regulates the assembly and tubulation of the Salmonella phagosome. SifA localizes to the phagosome and interacts with the membrane via its prenylated tail. SifA is a structural homologue of another bacterial effector that acts as a GTP-exchange factor for Rho family GTPases and can bind GDP-RhoA. When coexpressed with a bacterial lipase that is activated by RhoA, SifA can induce tubulation of mammalian endosomes. In an effort to develop a genetic system to study SifA function, we expressed SifA and characterized its activity in yeast. GFP-SifA predominantly localized to yeast peroxisomal membranes. Under peroxisome-inducing conditions, GFP-SifA reduced the number of free peroxisomes and promoted the formation of large peroxisomes with membrane invaginations. GFP-SifA activity depended on the recruitment to peroxisomes of wild-type Rho1p and Pex25p, a receptor for Rho1p. GFP-SifA could also rescue the actin organization defects in pex25Δ and rho1 mutants, suggesting that SifA may recruit and potentiate Rho1p activity. We reexamined the distribution of GFP-SifA in mammalian cells and found the majority colocalizing with LAMP1-positive compartment and not with the peroxisomal marker PMP70. Together, these data suggest that SifA may use a similar mode of action via Rho proteins to alter yeast peroxisomal and mammalian endosomal membranes. Further definition of SifA activity on yeast peroxisomes could provide more insight into its role in regulating host membrane dynamics and small GTPases.     

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.     

Rat liver peroxisomes after fibrate treatment. A survey using quantitative mass spectrometry

Fibrates are known to induce peroxisome proliferation and the expression of peroxisomal beta-oxidation enzymes. To analyze fibrate-induced changes of complex metabolic networks, we have compared the proteome of rat liver peroxisomes from control and bezafibrate-treated rats. Highly purified peroxisomes were subfractionated, and the proteins of the matrix, peripheral, and integral membrane subfractions thus obtained were analyzed by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry after labeling of tryptic peptides with the iTRAQ reagent. By means of this quantitative technique, we were able to identify 134 individual proteins, covering most of the known peroxisomal proteome. Ten predicted new open reading frames were verified by cDNA cloning, and seven of them could be localized to peroxisomes by immunocytochemistry. Moreover, quantitative mass spectrometry substantiated the induction of most of the known peroxisome proliferator-activated receptor alpha-regulated peroxisomal proteins upon treatment with bezafibrate, documenting the suitability of the iTRAQ procedure in larger scale experiments. However, not all proteins reacted to a similar extent but exerted a fibrate-specific induction scheme showing the variability of peroxisome proliferator-activated receptoralpha-transmitted responses to specific ligands. In view of our data, rat hepatic peroxisomes are apparently not specialized to sequester very long chain fatty acids (C22-C26) but rather metabolize preferentially long chain fatty acids (C16-18).     

In-Depth Proteome Analysis of Arabidopsis Leaf Peroxisomes Combined with in Vivo Subcellular Targeting Verification Indicates Novel Metabolic and Regulatory Functions of Peroxisomes

Peroxisomes are metabolically diverse organelles with essential roles in plant development. The major protein constituents of plant peroxisomes are well characterized, whereas only a few low-abundance and regulatory proteins have been reported to date. We performed an in-depth proteome analysis of Arabidopsis (Arabidopsis thaliana) leaf peroxisomes using one-dimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry. We detected 65 established plant peroxisomal proteins, 30 proteins whose association with Arabidopsis peroxisomes had been previously demonstrated only by proteomic data, and 55 putative novel proteins of peroxisomes. We subsequently tested the subcellular targeting of yellow fluorescent protein fusions for selected proteins and confirmed the peroxisomal localization for 12 proteins containing predicted peroxisome targeting signals type 1 or 2 (PTS1/2), three proteins carrying PTS-related peptides, and four proteins that lack conventional targeting signals. We thereby established the tripeptides SLM> and SKV> (where > indicates the stop codon) as new PTS1s and the nonapeptide RVx₅HF as a putative new PTS2. The 19 peroxisomal proteins conclusively identified from this study potentially carry out novel metabolic and regulatory functions of peroxisomes. Thus, this study represents an important step toward defining the complete plant peroxisomal proteome.     

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.     

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.     

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.     

Evaluation of the role of the endoplasmic reticulum-Golgi transit in the biogenesis of peroxisomal membrane proteins in wild type and peroxisome biogenesis mutant CHO cells

Peroxisomes are thought to be formed by division of pre-existing peroxisomes after the import of newly synthesized proteins. However, it has been recently suggested that the endoplasmic reticulum (ER) provides an alternative de novo mechanism for peroxisome biogenesis in some cells. To test a possible role of the ER-Golgi transit in peroxisome biogenesis in mammalian cells, we evaluated the biogenesis of three peroxisomal membrane proteins (PMPs): ALDRP (adrenoleukodystrophy related protein), PMP70 and Pex3p in CHO cells. We constructed chimeric genes encoding these PMPs and green fluorescent protein (GFP), and transiently transfected them to wild type and mutant CHO cells, in which normal peroxisomes were replaced by peroxisomal membrane ghosts. The expressed proteins were targeted to peroxisomes and peroxisomal ghosts correctly in the presence or absence of Brefeldin A (BFA), a drug known to block the ER-Golgi transit. Furthermore, low temperature did not disturb the targeting of Pex3p-GFP to peroxisomes. We also constructed two chimeric proteins of PMPs containing an ER retention signal "DEKKMP": GFP-ALDRP-DEKKMP and myc- Pex3p-DEKKMP. These proteins were mostly targeted to peroxisomes. No colocalization with an ER maker was found. These results suggest that the classical ER-Golgi pathway does not play a major role in the biogenesis of mammalian PMPs.     

Going against the flow: A case for peroxisomal protein export

Peroxisomes play a crucial role in regulating cellular metabolism, providing compartments where metabolic pathways can be contained and controlled. Their importance is underlined by the developmental brain disorders caused by peroxisome malfunction, while disturbances in peroxisome function also contribute to ageing. As peroxisomes do not contain DNA, they rely on an active transport system to obtain the full quota of proteins required for function. Organelle protein transport however, is rarely a one-way process and exciting recent data have demonstrated that peroxisomes can selectively export membrane and matrix proteins to fulfil specific functions. This review will summarise the current knowledge on peroxisomal membrane and matrix protein export, discussing the mechanisms underlying export as well as the role of peroxisomal protein export in peroxisomal and cellular function.     

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.