Yarrowia lipolytica Pex20p, Saccharomyces cerevisiae Pex18p/Pex21p and mammalian Pex5pL fulfil a common function in the early steps of the peroxisomal PTS2 import pathway.

Import of peroxisomal matrix proteins is essential for peroxisome biogenesis. Genetic and biochemical studies using a variety of different model systems have led to the discovery of 23 PEX genes required for this process. Although it is generally believed that, in contrast to mitochondria and chloroplasts, translocation of proteins into peroxisomes involves a receptor cycle, there are reported differences of an evolutionary conservation of this cycle either with respect to the components or the steps involved in different organisms. We show here that the early steps of protein import into peroxisomes exhibit a greater similarity than was thought previously to be the case. Pex20p of Yarrowia lipolytica, Pex18p and Pex21p of Saccharomyces cerevisiae and mammalian Pex5pL fulfil a common function in the PTS2 pathway of their respective organisms. These non-orthologous proteins possess a conserved sequence region that most likely represents a common PTS2-receptor binding site and di-aromatic pentapeptide motifs that could be involved in binding of the putative docking proteins. We propose that not necessarily the same proteins but functional modules of them are conserved in the early steps of peroxisomal protein import.

The diversity of organelle protein import mechanisms.

It was once believed that common mechanisms underpin the transfer of proteins across the membrane systems of organelles such as mitochondria, chloroplasts and peroxisomes. Now that many of the core components of the translocases have been indentified, results discussed at a recent conference [Max-Delbrück-Centrum Symposium "Protein Transport and Stability"; Berlin, Germany; 21-26 March 2001. Organized by Thomas Sommer and Enno Hartmann.] stress just how diverse the mechanisms of transport into these organelles really are.

Peroxisomes: the extended shuttle to the peroxisome matrix.

A recent study indicates that protein import into the peroxisomal matrix occurs by a possibly unique mechanism involving the shuttling of cargo receptors into and out of the organelles.

The di-aromatic pentapeptide repeats of the human peroxisome import receptor PEX5 are separate high affinity binding sites for the peroxisomal membrane protein PEX14.

PEX5 functions as a mobile import receptor for peroxisomal matrix proteins with a peroxisomal targeting signal 1 (PTS1). A critical step within the PTS1-import pathway is the interaction between PEX5 and the peroxisome membrane-associated protein PEX14. Based on two-hybrid analyses in mammalian cells and complementary in vitro binding assays, we demonstrate that the evolutionarily conserved pentapeptide repeat motifs, WX(E/D/Q/A/S)(E/D/Q)(F/Y), in PEX5 bind to PEX14 with high affinity. The results obtained indicate that each of the seven di-aromatic pentapeptides of human PEX5 interacts separately at the same binding site in the N terminus of PEX14 with equilibrium dissociation constants in the low nanomolar range. Mutational analysis of the PEX14-binding motifs reveals that the conserved aromatic amino acids at position 1 or 5 are essential for high affinity binding. We propose that the side chains of the aromatic amino acids are in close proximity as part of an amphipathic alpha-helix and together form hydrophobic anchors for binding PEX5 to individual PEX14 molecules.

Disorders of peroxisome biogenesis due to mutations in PEX1: phenotypes and PEX1 protein levels.

Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD) are clinically overlapping syndromes, collectively called "peroxisome biogenesis disorders" (PBDs), with clinical features being most severe in ZS and least pronounced in IRD. Inheritance of these disorders is autosomal recessive. The peroxisome biogenesis disorders are genetically heterogeneous, having at least 12 different complementation groups (CGs). The gene affected in CG1 is PEX1. Approximately 65% of the patients with PBD harbor mutations in PEX1. In the present study, we used SSCP analysis to evaluate a series of patients belonging to CG1 for mutations in PEX1 and studied phenotype-genotype correlations. A complete lack of PEX1 protein was found to be associated with severe ZS; however, residual amounts of PEX1 protein were found in patients with the milder phenotypes, NALD and IRD. The majority of these latter patients carried at least one copy of the common G843D allele. When patient fibroblasts harboring this allele were grown at 30 degrees C, a two- to threefold increase in PEX1 protein levels was observed, associated with a recovery of peroxisomal function. This suggests that the G843D missense mutation results in a misfolded protein, which is more stable at lower temperatures. We conclude that the search for the factors and/or mechanisms that determine the stability of mutant PEX1 protein by high-throughput procedures will be a first step in the development of therapeutic strategies for patients with mild PBDs.

Pex12p of Saccharomyces cerevisiae is a component of a multi-protein complex essential for peroxisomal matrix protein import.

We have isolated the Saccharomyces cerevisiae pex12-1 mutant from a screen to identify mutants defective in peroxisome biogenesis. The pex12delta deletion strain fails to import peroxisomal matrix proteins through both the PTS1 and PTS2 pathway. The PEX12 gene was cloned by functional complementation of the pex12-1 mutant strain and encodes a polypeptide of 399 amino acids. ScPex12p is orthologous to Pex12 proteins from other species and like its orthologues, S. cerevisiae Pex12p contains a degenerate RING finger domain of the C3HC4 type in its essential carboxy-terminus. Localization studies demonstrate that Pex12p is an integral peroxisomal membrane protein, with its NH2-terminus facing the peroxisomal lumen and with its COOH-terminus facing the cytosol. Pex12p-deficient cells retain particular structures that contain peroxisomal membrane proteins consistent with the existence of peroxisomal membrane remnants ("ghosts") in pex12A null mutant cells. This finding indicates that pex12delta cells are not impaired in peroxisomal membrane biogenesis. In immunoisolation experiments Pex12p was co-purified with the RING finger protein Pex10p, the PTS1 receptor Pex5p and the docking proteins for the PTS1 and the PTS2 receptor at the peroxisomal membrane, Pex13p and Pex14p. Furthermore, two-hybrid experiments suggest that the two RING finger domains are sufficient for the Pex10p-Pex12p interaction. Our results suggest that Pex12p is a component of the peroxisomal translocation machinery for matrix proteins.

Mitochondrial protein import motor: the ATPase domain of matrix Hsp70 is crucial for binding to Tim44, while the peptide binding domain and the carboxy-terminal segment play a stimulatory role.

The import motor for preproteins that are targeted into the mitochondrial matrix consists of the matrix heat shock protein Hsp70 (mtHsp70) and the translocase subunit Tim44 of the inner membrane. mtHsp70 interacts with Tim44 in an ATP-dependent reaction cycle, binds to preproteins in transit, and drives their translocation into the matrix. While different functional mechanisms are discussed for the mtHsp70-Tim44 machinery, little is known about the actual mode of interaction of both proteins. Here, we have addressed which of the three Hsp70 regions, the ATPase domain, the peptide binding domain, or the carboxy-terminal segment, are required for the interaction with Tim44. By two independent means, a two-hybrid system and coprecipitation of mtHsp70 constructs imported into mitochondria, we show that the ATPase domain interacts with Tim44, although with a reduced efficiency compared to the full-length mtHsp70. The interaction of the ATPase domain with Tim44 is ATP sensitive. The peptide binding domain and carboxy-terminal segment are unable to bind to Tim44 in the absence of the ATPase domain, but both regions enhance the interaction with Tim44 in the presence of the ATPase domain. We conclude that the ATPase domain of mtHsp70 is essential for and directly interacts with Tim44, clearly separating the mtHsp70-Tim44 interaction from the mtHsp70-substrate interaction.

Pex8p, an intraperoxisomal peroxin of Saccharomyces cerevisiae required for protein transport into peroxisomes binds the PTS1 receptor pex5p.

We report the characterization of ScPex8p, which is essential for peroxisomal biogenesis in Saccharomyces cerevisiae. Cells lacking Pex8p are characterized by the presence of peroxisomal membrane ghosts and mislocalization of peroxisomal matrix proteins of the PTS1 and PTS2 variety to the cytosol. Pex8p is tightly associated with the lumenal face of the peroxisomal membrane. Consistent with its intraperoxisomal localization, Pex8p contains a peroxisomal targeting signal 1, and it interacts with the PTS1 receptor Pex5p. However, the Pex5p/Pex8p association is also observed upon deletion of the PTS1 of Pex8p, suggesting that Pex8p contains a second binding site for Pex5p. The pex8Delta mutant phenotype and the observed PTS1-independent interaction with the PTS1 receptor suggest that Pex8p is involved in protein import into the peroxisomal matrix. In pex8Delta cells, the PTS1 and PTS2 receptor still associate with membrane bound components of the protein import machinery, supporting the assumption that the Pex8p function in protein translocation follows the docking event.

Identification and characterization of the human peroxin PEX3.

The biogenesis of peroxisomes requires the interaction of several peroxins, encoded by PEX genes and is well conserved between yeast and humans. We have cloned the human cDNA of PEX3 based on its homology to different yeast PEX3 genes. The deduced peroxin HsPEX3 is a peroxisomal membrane protein with a calculated molecular mass of 42.1 kDa. We created N- and C-terminal tagged PEX3 to assay its topology at the peroxisomal membrane by immunofluorescence microscopy. Our results and the one predicted transmembrane spanning region are in line with the assumption that H sPEX3 is an integral peroxisomal membrane protein with the N-terminus inside the peroxisome and the C-terminus facing the cytoplasm. The farnesylated peroxisomal membrane protein PEX19 interacts with HsPEX3 in a mammalian two-hybrid assay in human fibroblasts. The physical interaction could be confirmed by coimmunoprecipitation of the two in vitro transcribed and translated proteins. To address the targeting of PEX3 to the peroxisomal membrane, the expression of different N- and C-terminal PEX3 truncations fused to green fluorescent protein (GFP) was investigated in human fibroblasts. The N-terminal 33 amino acids of PEX3 were necessary and sufficient to direct the reporter protein GFP to peroxisomes and seemed to be integrated into the peroxisomal membrane. The expression of a 1-16 PEX3-GFP fusion protein did not result in a peroxisomal localization, but interestingly, this and several other truncated PEX3 fusion proteins were also localized to tubular and/or vesicular structures representing mitochondria.

Involvement of Pex13p in Pex14p localization and peroxisomal targeting signal 2-dependent protein import into peroxisomes.

Pex13p is the putative docking protein for peroxisomal targeting signal 1 (PTS1)-dependent protein import into peroxisomes. Pex14p interacts with both the PTS1- and PTS2-receptor and may represent the point of convergence of the PTS1- and PTS2-dependent protein import pathways. We report the involvement of Pex13p in peroxisomal import of PTS2-containing proteins. Like Pex14p, Pex13p not only interacts with the PTS1-receptor Pex5p, but also with the PTS2-receptor Pex7p; however, this association may be direct or indirect. In support of distinct peroxisomal binding sites for Pex7p, the Pex7p/Pex13p and Pex7p/ Pex14p complexes can form independently. Genetic evidence for the interaction of Pex7p and Pex13p is provided by the observation that overexpression of Pex13p suppresses a loss of function mutant of Pex7p. Accordingly, we conclude that Pex7p and Pex13p functionally interact during PTS2-dependent protein import into peroxisomes. NH2-terminal regions of Pex13p are required for its interaction with the PTS2-receptor while the COOH-terminal SH3 domain alone is sufficient to mediate its interaction with the PTS1-receptor. Reinvestigation of the topology revealed both termini of Pex13p to be oriented towards the cytosol. We also found Pex13p to be required for peroxisomal association of Pex14p, yet the SH3 domain of Pex13p may not provide the only binding site for Pex14p at the peroxisomal membrane.

Recombinant human peroxisomal targeting signal receptor PEX5. Structural basis for interaction of PEX5 with PEX14.

Import of matrix proteins into peroxisomes requires two targeting signal-specific import receptors, Pex5p and Pex7p, and their binding partners at the peroxisomal membrane, Pex13p and Pex14p. Several constructs of human PEX5 have been overexpressed and purified by affinity chromatography in order to determine functionally important interactions and provide initial structural information. Sizing chromatography and electron microscopy suggest that the two isoforms of the human PTS1 receptor, PEX5L and PEX5S, form homotetramers. Surface plasmon resonance analysis indicates that PEX5 binds to the N-terminal fragment of PEX14-(1-78) with a very high affinity in the low nanomolar range. Stable complexes between recombinant PEX14-(1-78) and both the full-length and truncated versions of PEX5 were formed in vitro. Analysis of these complexes revealed that PEX5 possesses multiple binding sites for PEX14, which appear to be distributed throughout its N-terminal half. Coincidentally, this part of the molecule is also responsible for oligomerization, whereas the C-terminal half with its seven tetratricopeptide repeats has been reported to bind PTS1-proteins. A pentapeptide motif that is reiterated seven times in PEX5 is proposed as a determinant for the interaction with PEX14.

Identification and characterization of the human orthologue of yeast Pex14p.

Pex14p is a central component of the peroxisomal protein import machinery, which has been suggested to provide the point of convergence for PTS1- and PTS2-dependent protein import in yeast cells. Here we describe the identification of a human peroxisome-associated protein (HsPex14p) which shows significant similarity to the yeast Pex14p. HsPex14p is a carbonate-resistant peroxisomal membrane protein with its C terminus exposed to the cytosol. The N terminus of the protein is not accessible to exogenously added antibodies or protease and thus might protrude into the peroxisomal lumen. HsPex14p overexpression leads to the decoration of tubular structures and mislocalization of peroxisomal catalase to the cytosol. HsPex14p binds the cytosolic receptor for the peroxisomal targeting signal 1 (PTS1), a result consistent with a function as a membrane receptor in peroxisomal protein import. Homo-oligomerization of HsPex14p or interaction of the protein with the PTS2-receptor or HsPex13p was not observed. This distinguishes the human Pex14p from its counterpart in yeast cells and thus supports recent data suggesting that not all aspects of peroxisomal protein import are conserved between yeasts and humans. The role of HsPex14p in mammalian peroxisome biogenesis makes HsPEX14 a candidate PBD gene for being responsible for an unrecognized complementation group of human peroxisome biogenesis disorders.

Peroxisome biogenesis: back to the endoplasmic reticulum?

Proteins are targeted to the membrane and matrix of peroxisomes by distinct pathways. Recent observations suggest a further route: a subset of peroxisomal membrane proteins might be targeted first to the endoplasmic reticulum, and from there to peroxisomes by vesicle-mediated transport.

Pex17p of Saccharomyces cerevisiae is a novel peroxin and component of the peroxisomal protein translocation machinery.

The Saccharomyces cerevisiae pex17-1 mutant was isolated from a screen to identify mutants defective in peroxisome biogenesis. pex17-1 and pex17 null mutants fail to import matrix proteins into peroxisomes via both PTS1- and PTS2-dependent pathways. The PEX17 gene (formerly PAS9; Albertini, M., P. Rehling, R. Erdmann, W. Girzalsky, J.A.K.W. Kiel, M. Veenhuis, and W.-H Kunau. 1997. Cell. 89:83-92) encodes a polypeptide of 199 amino acids with one predicted membrane spanning region and two putative coiled-coil structures. However, localization studies demonstrate that Pex17p is a peripheral membrane protein located at the surface of peroxisomes. Particulate structures containing the peroxisomal integral membrane proteins Pex3p and Pex11p are evident in pex17 mutant cells, indicating the existence of peroxisomal remnants ("ghosts"). This finding suggests that pex17 null mutant cells are not impaired in peroxisomal membrane biogenesis. Two-hybrid studies showed that Pex17p directly binds to Pex14p, the recently proposed point of convergence for the two peroxisomal targeting signal (PTS)-dependent import pathways, and indirectly to Pex5p, the PTS1 receptor. The latter interaction requires Pex14p, indicating the potential of these three peroxins to form a trimeric complex. This conclusion is supported by immunoprecipitation experiments showing that Pex14p and Pex17p coprecipitate with both PTS receptors in the absence of Pex13p. From these and other studies we conclude that Pex17p, in addition to Pex13p and Pex14p, is the third identified component of the peroxisomal translocation machinery.

Pex19p, a farnesylated protein essential for peroxisome biogenesis.

We report the identification and molecular characterization of Pex19p, an oleic acid-inducible, farnesylated protein of 39.7 kDa that is essential for peroxisome biogenesis in Saccharomyces cerevisiae. Cells lacking Pex19p are characterized by the absence of morphologically detectable peroxisomes and mislocalization of peroxisomal matrix proteins to the cytosol. The human HK33 gene product was identified as the putative human ortholog of Pex19p. Evidence is provided that farnesylation of Pex19p takes place at the cysteine of the C-terminal CKQQ amino acid sequence. Farnesylation of Pex19p was shown to be essential for the proper function of the protein in peroxisome biogenesis. Pex19p was shown to interact with Pex3p in vivo, and this interaction required farnesylation of Pex19p.

Peroxisome biogenesis: from yeast to man.

A major current issue in studies of peroxisome biogenesis is how proteins are imported into the organelle or inserted into its membrane. Recent studies indicate that these two processes use independent pathways. Both appear to have unexpected properties. Matrix proteins can be imported in an oligomeric form which might be facilitated by cycling receptors, whereas insertion of at least some peroxisomal membrane proteins seems to involve the endoplasmic reticulum.

Human PEX1 is mutated in complementation group 1 of the peroxisome biogenesis disorders.

Human peroxisome biogenesis disorders (PBDs) are a group of genetically heterogeneous autosomal-recessive disease caused by mutations in PEX genes that encode peroxins, proteins required for peroxisome biogenesis. These lethal diseases include Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum's disease (IRD), three phenotypes now thought to represent a continuum of clinical features that are most severe in ZS, milder in NALD and least severe in IRD2. At least eleven PBD complementation groups have been identified by somatic-cell hybridization analysis compared to the eighteen PEX complementation groups that have been found in yeast. We have cloned the human PEX1 gene encoding a 147-kD member of the AAA protein family (ATPases associated with diverse cellular activities), which is the putative orthologue of Saccharomyces cerevisiae Pex1p (ScPex1p). Human PEX1 has been identified by computer-based 'homology probing' using the ScPex1p sequence to screen databases of expressed sequence tags (dbEST) for human cDNA clones. Expression of PEX1 rescued the cells from the biogenesis defect in human fibroblasts of complementation group 1 (CG1), the largest PBD complementation group. We show that PEX1 is mutated in CG1 patients.

The 1.8 A crystal structure of the dimeric peroxisomal 3-ketoacyl-CoA thiolase of Saccharomyces cerevisiae: implications for substrate binding and reaction mechanism.

The dimeric, peroxisomal 3-ketoacyl-CoA thiolase catalyses the conversion of 3-ketoacyl-CoA into acyl-CoA, which is shorter by two carbon atoms. This reaction is the last step of the beta-oxidation pathway. The crystal structure of unliganded peroxisomal thiolase of the yeast Saccharomyces cerevisiae has been refined at 1.8 A resolution. An unusual feature of this structure is the presence of two helices, completely buried in the dimer and sandwiched between two beta-sheets. The analysis of the structure shows that the sequences of these helices are not hydrophobic, but generate two amphipathic helices. The helix in the N-terminal domain exposes the polar side-chains to a cavity at the dimer interface, filled with structured water molecules. The central helix in the C-terminal domain exposes its polar residues to an interior polar pocket. The refined structure has also been used to predict the mode of binding of the substrate molecule acetoacetyl-CoA, as well as the reaction mechanism. From previous studies it is known that Cys125, His375 and Cys403 are important catalytic residues. In the proposed model the acetoacetyl group fits near the two catalytic cysteine residues, such that the oxygen atoms point towards the protein interior. The distance between SG(Cys125) and C3(acetoacetyl-CoA) is 3.7 A. The O2 atom of the docked acetoacetyl group makes a hydrogen bond to N(Gly405), which would favour the formation of the covalent bond between SG(Cys125) and C3(acetoacetyl-CoA) of the intermediate complex of the two-step reaction. The CoA moiety is proposed to bind in a groove on the surface of the protein molecule. Most of the interactions of the CoA molecule are with atoms of the loop domain. The three phosphate groups of the CoA moiety are predicted to interact with side-chains of lysine and arginine residues, which are conserved in the dimeric thiolases.

Genomic organization and molecular characterization of a gene encoding HsPXF, a human peroxisomal farnesylated protein.

A protein modification essential for the cellular sorting of many biologically relevant proteins is the covalent attachment of prenyl lipids by specific transferases. Isoprenylation is known to render protein domains hydrophobic, thereby facilitating the interaction with lipid bilayers and/or membrane proteins. The target for the modification with farnesyl groups is the COOH-terminal sequence CaaX. Among the variety of farnesylated proteins the only one reported so far to be located to peroxisomes is the 37-kDa peroxisomal farnesylated hamster protein PxF. Recently we published data on the cDNA of the human gene HK33 (A. Braun et al., 1994, Gene 146: 291-295), which was revealed to be the human ortholog of PxF and was consequently renamed HsPXF. The genomic structure, molecular characterization, and evolutionary conservation of HsPXF are described herein. The exact location of the gene was defined as chromosome 1q22. The gene spans a region of approximately 9 kb, containing eight exons and seven introns. The 5' upstream region showed two potential Sp1-binding sites and an Alu repetitive sequence. Luciferase reporter activating capacity confirmed the presumed promoter activity of this region. On the transcriptional level, we detected four splice variants originating either from exon skipping or from alternative splicing events. For the HsPXF protein, a carboxyterminal farnesylation at cysteine residues was demonstrated. Through the use of HsPXF-specific antibodies, the protein was shown to be attached to the outer surface of peroxisomes. This localization together with the similarity to a peroxisomal assembly protein from Saccharomyces cerevisiae suggests HsPXF is involved in the process of peroxisomal biogenesis or assembly.

Pex14p, a peroxisomal membrane protein binding both receptors of the two PTS-dependent import pathways.

Pex14p, an S. cerevisiae peroxin, is attached to the outer face of the peroxisomal membrane and is a component of the protein import machinery. Pex14p interacts with both the PTS1 and PTS2 receptors. It is the only known peroxisomal membrane protein that binds the PTS2 receptor and might thus mediate the membrane docking event of PTS2-dependent protein import. These results suggest that the two import pathways overlap and, furthermore, that Pex14p represents the point of convergence. Pex14p also interacts with two other membrane-bound peroxins including Pex13p, another binding protein for the PTS1 receptor. The data presented here are consistent with the idea of a common translocation machinery for both PTS-dependent protein import pathways in the peroxisomal membrane.