Peroxisomal disorders I: biochemistry and genetics of peroxisome biogenesis disorders

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1 Clin Genet 2004: 67: Copyright # Blackwell Munksgaard 2004 Printed in Singapore. All rights reserved CLINICAL GENETICS doi: /j x Mini Review Peroxisomal disorders I: biochemistry and genetics of peroxisome biogenesis disorders Wanders RJA, Waterham HR. Peroxisomal disorders I: biochemistry and genetics of peroxisome biogenesis disorders. Clin Genet 2004: 67: # Blackwell Munksgaard, 2004 The peroxisomal disorders represent a group of genetic diseases in humans in which there is an impairment in one or more peroxisomal functions. The peroxisomal disorders are usually subdivided into two subgroups including (i) the peroxisome biogenesis disorders (PBDs) and (ii) the single peroxisomal (enzyme-) protein deficiencies. The PBD group is comprised of four different disorders including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantile Refsum s disease (IRD), and rhizomelic chondrodysplasia punctata (RCDP). ZS, NALD, and IRD are clearly distinct from RCDP and are usually referred to as the Zellweger spectrum with ZS being the most severe and NALD and IRD the less severe disorders. Studies in the late 1980s had already shown that the PBD group is genetically heterogeneous with at least 12 distinct genetic groups as concluded from complementation studies. Thanks to the much improved knowledge about peroxisome biogenesis notably in yeasts and the successful extrapolation of this knowledge to humans, the genes responsible for all these complementation groups have been identified making molecular diagnosis of PBD patients feasible now. It is the purpose of this review to describe the current stage of knowledge about the clinical, biochemical, cellular, and molecular aspects of PBDs, and to provide guidelines for the post- and prenatal diagnosis of PBDs. Less progress has been made with respect to the pathophysiology and therapy of PBDs. The increasing availability of mouse models for these disorders is a major step forward in this respect. RJA Wanders and HR Waterham Department of Pediatrics, Academic Medical Centre, Emma Children s Hospital, University of Amsterdam, and Department of Clinical Chemistry, Laboratory Genetic Metabolic Diseases, Amsterdam, The Netherlands Key words: fatty acids genetics inborn errors peroxisome biogenesis peroxisomes Corresponding author: Prof. Dr Ronald J. A. Wanders, Lab Genetic Metabolic Diseases, F0-224, Academic Medical Centre, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Tel.: þ ; fax: þ ; r.j.wanders@amc.uva.nl Received 7 May 2004, revised and accepted for publication 22 June 2004 Zellweger syndrome (ZS) is the prototype of the group of peroxisomal disorders and was first described in the 1960s in two pairs of sibs, showing a series of abnormalities including craniofacial, hepatological, ocular, and skeletal aberrations. At about the same time, De Duve and coworkers performed systematic studies in which rat liver homogenates were subjected to differential and density gradient centrifugation. These studies led to the identification of a new organelle containing a number of H 2 O 2 -generating oxidases and catalase which decomposes H 2 O 2 to O 2 and H 2 O. The connection between ZS and peroxisomes first became apparent in 1973 when Goldfischer et al. (1) reported the absence of morphologically identifiable peroxisomes in hepatocytes and kidney tubule cells of Zellweger patients. At that time, however, virtually nothing was known about peroxisomes and it took another 10 years before the true significance of peroxisomes for human physiology started to become clear, thanks to two key observations in Zellweger patients. First, Brown et al. (2) discovered distinct abnormalities in the fatty acid profile of plasma from Zellweger patients with markedly elevated levels of the very-longchain fatty acids (VLCFAs) C24:0 and C26:0, whereas normal levels were found for the other fatty acids including long-chain fatty acids like palmitic, oleic, and linoleic acid. At that time, peroxisomes were already known to contain a fatty acid beta-oxidation system, just like mitochondria, but the function of this system had remained obscure. The findings by Brown et al. (2) suggested that peroxisomes are the site of beta-oxidation of VLCFAs, which was soon established experimentally (3). The second major 107

2 Wanders and Waterham discovery demonstrating the crucial role of peroxisomes in humans appeared 1 year later when Heymans et al. (4) reported the deficiency of plasmalogens, a special type of phospholipids belonging to the group of ether-linked phospholipids, in tissues from Zellweger patients. Since then, much has been learned about the metabolic role of peroxisomes and many different functions of peroxisomes have been identified. In addition, many of the enzymes involved in the different metabolic pathways within peroxisomes have been characterized, purified, and their respective cdnas and genes cloned. Parallel to this work, the essential details of peroxisome biogenesis have been worked out and many of the genes, coding for proteins essential for peroxisome biogenesis, have been identified. Thanks to this explosion of new information, enormous progress has been made with respect to the identification of new peroxisomal disorders followed by resolution of the underlying defects. At present, the group of peroxisomal disorders comprises 17 well-defined disorders, which are subdivided into two groups including (i) the peroxisome biogenesis disorders (PBDs) and (ii) the single peroxisomal (enzyme-) protein deficiencies. This review is focused on the first group of disorders, the PBDs (Table 1), and we will begin by discussing what is known about the different PBDs. The peroxisome biogenesis disorders: a clinically and genetically heterogeneous group of disorders The PBD group is comprised of four different disorders including ZS, neonatal adrenoleukodystrophy (NALD), infantile Refsum s disease (IRD), and rhizomelic chondrodysplasia punctata (RCDP). ZS, NALD, and IRD are clearly distinct from RCDP and are nowadays usually referred to as the Zellweger spectrum with ZS being the most severe and NALD and IRD less severe disorders. ZS is generally considered as the prototype of the PBD group. ZS is dominated by: (i) the typical craniofacial dysmorphism including a high forehead, large anterior fontanel, hypoplastic supraorbital ridges, epicanthal folds, and deformed earlobes, and (ii) profound neurological abnormalities. ZS children show severe psychomotor retardation, profound hypotonia, neonatal seizures, glaucoma, retinal degeneration, and impaired hearing. There is usually calcific stippling of the epiphyses and small renal cysts. Brain abnormalities in ZS include not only cortical dysplasia and neuronal heterotopia but also regressive changes. There is dysmyelination rather than demyelination. Patients with NALD have hypotonia and seizures, may have polymicrogyria, progressive white matter disease, and usually die in late infancy. Patients with IRD may have external features reminiscent of ZS but do not show disordered neuronal migration and no progressive white matter disease. Their cognitive and motor development varies between severe global handicaps and moderate learning disabilities with deafness and visual impairment due to retinopathy. Their survival is variable. Most patients with IRD reach childhood and some even reach adulthood. Clinical distinction between the different PBD phenotypes is not very well defined. Common to all three are liver disease, variable neurodevelopmental delay, retinopathy, and perceptive deafness with onset in the first months of life. RCDP is clinically quite different from ZS, NALD, and IRD and characterized by a disproportionally short stature primarily affecting the proximal parts of the extremities, typical facial appearance, including a broad nasal bridge, epicanthus, high arched palate, dysplastic external ears, micrognathia, congenital contractures, characteristic ocular involvement, dwarfism, and severe mental retardation with spasticity. Most RCDP patients die in the first decade of life. ZS, NALD, IRD, and RCDP have been found to be genetically heterogeneous as concluded from complementation studies as discussed later in this review. The molecular defects underlying these different complementation groups (CGs) have been resolved in recent years. Two different strategies have been very rewarding in the identification of these mutant genes, which includes (i) homology probing, making use of the information from different yeast mutants, and (ii) functional complementation analysis based on the generation of peroxisome-deficient Chinese Table 1. The peroxisome biogenesis disorders Number Disorder Abbreviation Protein involved Gene Chromosome MIM 1 Zellweger syndrome ZS Peroxins PEX-genes Multiple loci Neonatal adrenoleukodystrophy NALD Peroxins PEX-genes Multiple loci Infantile Refsum s disease IRD Peroxins PEX-genes Multiple loci Rhizomelic chondrodysplasia punctata type 1 RCDP Type 1 Pex7p PEX7 6q21 q

3 Peroxisomal disorders hamster ovary (CHO) cells. We will proceed by describing the current stage of knowledge about peroxisome biogenesis. Peroxisome biogenisis: general aspects Peroxisomal proteins are all encoded by nuclear genes and translated on free polyribosomes as first shown for urate oxidase and catalase, two peroxisomal matrix proteins, by Goldmann and Blobel (5), and Robbi and Lazarow (6), respectively. Later studies have shown the same for peroxisomal membrane proteins (PMPs) (7). After synthesis on free polyribosomes, the newly made peroxisomal proteins are targeted to peroxisomes and then imported into pre-existing peroxisomes post-translationally, which implies that synthesis and import are sequential rather than simultaneous processes. In this way, peroxisomes get bigger which requires recruitment of phospholipids most likely from the endoplasmic reticulum (ER) to be incorporated into the peroxisomal membrane. Growth of peroxisomes may continue until a critical size is reached after which peroxisomes divide into two daughter peroxisomes that can then undergo the same cycle of events (Fig. 1a). The import of peroxisomal matrix and membrane proteins into peroxisomes is a multistep process involving recognition of the cargo protein by a receptor in the cytosol, docking of the receptor cargo complex at the peroxisomal membrane, translocation across the membrane, cargo release into the organelle, and receptor recycling. Correct targeting of peroxisomal matrix proteins is achieved via cis-acting sequences present in the primary peptide sequences, which are called peroxisomal targeting signals (PTSs). Most matrix proteins are equipped with a PTS type 1 (PTS1), which is a C-terminal serine-lysine-leucine- COOH (SKL) tripeptide, or a conservative variant thereof, like SHL in D-aminoacid oxidase, AKL in sterol carrier protein 2 (SCP2), etc (Table 2). A few matrix proteins are targeted via a different signal named PTS2, which is a 9-amino acid sequence located near the N-terminus with the amino acids in positions 1, 2, 8, and 9 being most important. The consensus PTS2 is R/K-L/V/ I-XXXXX-H/Q-A/L in which X is any amino acid. The PTS1 and PTS2 receptors have been Fig. 1. Original (a) and modified (b) model for peroxisome biogenesis. (a) The original growth-and-division model proposed by Lazarow and Fujiki (139) in which peroxisomes were thought to be autonomous organelles, which could not form de novo. (b) The modified model of Lazarow and Fujiki with peroxisomes now envisaged as semiautonomous organelles with the capacity to form de novo. 109

4 Wanders and Waterham Table 2. List of bona fide peroxisomal (enzyme) proteins from humans and their PTS1 or PTS2 sequences Peroxisomal function (Enzyme) protein PTS1/PTS2 Targeting sequence Fatty acid b-oxidation Acyl-CoA oxidase 1 (straight chain) PTS1 SKL Acyl-CoA oxidase 2 (branched chain) PTS1 SKL Acyl-CoA oxidase 3 (pristanoyl-coa) PTS1 SKL L-bifunctional protein PTS1 SKL D-bifunctional protein PTS1 AKL 3-ketothiolase (straight chain) PTS2 RLQVVLGHL 3-ketothiolase (branched chain) PTS1 AKL 2-methylacyl-CoA racemase PTS1 (þmts) (K)ASL Carnitine acetyltransferase PTS1 AKL Carnitine octanoyltransferase PTS1 THL Acyl-CoA thioesterase PTS1 SKL Bile acid-coa: taurine/glycine conjugating enzyme PTS1 SQL 2,4-dienoylCoA reductase PTS1 AKL D2,D3-enoylCoA isomerase PTS1 SKL D 3,5, D 2,4 -dienoylcoa isomerase PTS1 SKL Very-long-chain acyl-coa synthetase (VLACS) PTS1 LKL Fatty acid a-oxidation PhytanoylCoA hydroxylase PTS2 RLQIVLGHL 2-hydroxyphytanoylCoA lyase PTS1 (R)SNM Etherphospholipid biosynthesis Dihydroxyacetonephosphate acyltransferase PTS1 AKL Alkyldihydroxyacetonephosphate synthase PTS2 RLVLSGHL Glyoxylate detoxification Alanine glyoxylate aminotransferase PTS1 KKL Pipecolic acid degradation L-pipecolate oxidase PTS1 AHL H 2 O 2 metabolism Catalase PTS1 (K)ANL Peroxiredoxin V PTS1 SQL Sterol carrier protein 2 PTS1 AKL D-aspartate oxidase PTS1 (K)SNL D-amino acid oxidase PTS1 SHL Hydroxyacid oxidase 3 PTS1 SRL Hydroxyacid oxidase 2 PTS1 SRL Hydroxyacid oxidase 1 (glycolate oxidase) PTS1 SKL Others 3-hydroxy-3-methyl glutarylcoa lyase PTS1 (þmts) CKL MalonylCoA decarboxylase PTS1 (þmts) SKL Isocitrate dehydrogenase PTS1 AKL PTS1, peroxisome-targeting signal type 1; PTS2, peroxisome-targeting signal type 2; MTS, mitochondrial targeting signal. In the right hand column, the different targeting sequences are shown using the single letter code for the various amino acids. cloned and characterized from different species. The former, Pex5p, is a tetratricopeptide (TPR) repeat protein, whereas the latter, Pex7p, is a WD40 repeat protein, to be discussed later. Similar to matrix proteins, PMPs are synthesized on free cytosolic ribosomes and targeted to the organelle by cis-acting targeting sequences (mpts). In contrast to the simple PTS1 and PTS2 sequences found in matrix proteins, PMPs are directed to peroxisomes via, as yet, less well-defined targeting signals, to be discussed later. Peroxisome biogenesis: de novo formation of peroxisomes or not? As discussed above, peroxisome biogenesis resembles that of mitochondria and chloroplasts, which is true although the details are entirely different. Indeed, protein translocation into peroxisomes differs markedly from that in mitochondria which threads unfolded polypeptide chains through a narrow channel, whereas peroxisomes can import folded and homo-oligomeric proteins (8), hetero-oligomers (9, 10), and even 4 9-nm gold beads (11). The transport of such large complexes somewhat resembles protein transport into the nucleus, but no such thing as a structure resembling the nuclear pore complex has ever been observed in the peroxisomal membrane. The concept that peroxisomes multiply by growth and division of pre-existing peroxisomes would make peroxisomes belong to the group of autonomousorganelleswithmitochondria, chloroplasts, and the endoplasmic reticulum as representatives. This would imply that peroxisomes cannot form de novo. Several experimental observations have been done suggesting that peroxisomes can form de novo, however. One of the main arguments in favor of de novo biogenesis of peroxisomes has been that cells, mutated in PEX3, PEX16, or PEX19, show no peroxisomal membrane structures (ghosts), whereas reintroduction of a wild-type copy of the mutant gene restores peroxisome formation. These findings have been interpreted as evidence for de novo synthesis of peroxisomes from some endomembrane compartment such as the ER (Fig. 1b). 110

5 Peroxisomal disorders Based on studies in the yeasts Yarrowia lipolytica and Hansenula polymorpha, it has been proposed that peroxisomes can be formed from small pre-peroxisomal vesicles derived from the ER in a process dependent on COPI and COPII, two coat proteins involved in vesicle transport processes. Studies in human fibroblasts, however, have shown that peroxisome biogenesis occurs independent of COPI and COPII (12, 13). Furthermore, studies by South et al. (14) in the yeast Saccharomyces cerevisiae suggest that protein traffic into the ER is not required to form peroxisomes. This was concluded from studies in which the protein entry into the ER was blocked by inactivation of the ER protein translocation factor, Sec61p, or its homolog, Ssh1p. These results argue against the ER as the site of de novo peroxisome formation. Furthermore, studies by Snyder et al. (15) and Hazra et al. (16) have provided compelling evidence against the dogma of the absence of peroxisomal structures in pex3d, pex16d, and pex19d mutants. Indeed, Snyder et al. (15) identified tiny peroxisomal vesicles and tubules in Pichia pastoris pex19d cells by deconvolution microscopy using an antibody recognizing endogenous Pex3p. In addition, Hazra et al. (16) reported the identification of vesicular and tubular, torpedo-shaped peroxisomal structures in P. pastoris pex3d cells and characterized these by isopyknic and flotation centrifugation. The jury is still out on the origin of peroxisomes, however, as emphasized by several very recent studies. Firstly, Geuze et al. (17) recently presented evidence of the involvement of the ER in peroxisome formation in mouse dendritic cells using electron microscopy, immunocytochemistry, and three-dimensional image reconstruction of peroxisomes and associated compartments. Additional support for the formation of peroxisomes from some endomembrane compartment has also come from studies by Faber et al. (18) who have shown that an N-terminal fragment of Pex3p expressed in H. polymorpha is associated with vesicular membrane structures that also contain Pex14p. Furthermore, these structures appeared to have the potential to develop into functional peroxisomes after introduction of full-length PEX3 and arise from the nuclear membrane. In conclusion, it remains to be established whether there are indeed two parallel pathways for peroxisome formation, one from pre-existing peroxisomes and a second de novo pathway, which allows peroxisome formation from some endomembrane compartment such as the ER. Peroxisome biogenesis: a closer look The realization that a simple organism like baker s yeast could be used to study peroxisome biogenesis and resolve the sorting and targeting of peroxisomal proteins to their correct destination, the peroxisome, has had a tremendous impact and explains for a large part why the pursuit of genes defect in PBD patients has been so fruitful in the last few years. The key to the application of genetics to the elucidation of the mechanism of peroxisome biogenesis and the identification of the proteins involved was the isolation of peroxisome-deficient mutants (pex mutants) from different yeast species and CHO mutants (19). Erdmann et al. (20) were the first to device a selection screen based on the notion that in yeast, peroxisomes are essential for growth on oleate. This follows logically from the fact that in yeast, fatty acids can only be oxidized in peroxisomes whereas in higher eukaryotes betaoxidation can occur both in peroxisomes and in mitochondria. S. cerevisiae cells were subjected to chemical mutagenesis and grown first on glucose agar plates followed by replica plating onto oleate agar plates to select for cells not growing on oleate (onu-mutants). Subsequently, cell fractionation studies were performed to eliminate mutants with no abnormalities in peroxisome biogenesis but a defect in the fatty acid beta-oxidation system. This approach resulted in a total of 12 different mutants that turned out to be peroxisome deficient. Similar screens have been set up for a variety of different yeast species including P. pastoris, H. polymorpha, and Y. lipolytica. Additional screens and selections, based on other approaches, have also been set up which together has led to the generation of a large series of peroxisome biogenesis mutants. Subsequent complementation of these mutants using yeast genomic libraries has resulted in the identification of a large number of genes involved in peroxisome biogenesis. Initially, these new genes were all given different names even within the same species (i.e. PAF, PAS, PEB, PER, and PAY genes). To simplify matters, all of these genes have been renamed as PEX genes (PEX1, PEX2, PEX, etc) and the products of these genes are called peroxins (Pex1p, Pex2p, Pex3p, etc). The peroxins were agreed to include all proteins involved in peroxisome biogenesis inclusive of peroxisome matrix protein import, membrane biogenesis, peroxisome proliferation, and peroxisome inheritance. In the original study of Erdmann et al. (20), 12 different S. cerevisiae mutants were identified in which peroxisome biogenesis was impaired. 111

6 Wanders and Waterham One by one the genes mutated in each of these so-called pas-mutants have been identified, of which the first one was described by Erdmann et al. in 1991 (21). The gene involved (PEX1) codes for a protein belonging to the family of triple A (AAA) ATPases, which are involved in the assembly, organization, and disassembly of protein complexes (22). The discovery of the first peroxisome biogenesis gene in S. cerevisiae was soon followed by reports from the same group describing the second (PEX3) (23) and third (PEX4) (24) S. cerevisiae PEX genes. In pex1d, pex3d, and pex4d cells, the import of PTS1 and PTS2 proteins is impaired, indicating that Pex1p, Pex3p, and Pex4p play an essential role in the import of matrix proteins. Later studies revealed that these mutants are different if the import of PMPs is studied. Indeed, pex1d and pex4d cells are still able to assemble their PMPs into membranes, whereas pex3d cells lack this property. Studies by Hettema et al. (25) in a series of 19 S. cerevisiae mutants have shown that the import of PTS1 and/or PTS2 proteins is impaired in all mutants except one (pex11d), whereas PMP import is normal in all these mutants except for the pex3d and pex19d mutants. These data are in line with the notion that Pex3p and Pex19p belong to a distinct group of peroxins required for the proper localization and stabilization of PMPs as discussed in the next section. With the recent identification of three PEX genes in the yeast S. cerevisiae, i.e. PEX 30, 31, and 32 (26), the total number of PEX genes now stands at 32 (Table 3). The complete set of 32 PEX genes can be subdivided into two groups in which group 1 includes those genes of which orthologs are found among most, if not all, peroxisome-containing species, whereas group 2 refers to those PEX genes which are only found in single organisms. Most of the PEX genes belong to group 1 with orthologs in different species. PEX genes belonging to group 2 are PEX18 and PEX21, which are only found in S. cerevisiae (27), and PEX20 which is only found in Y. lipolytica (28) and Neurospora crassa (29). These results indicate that the principal features of peroxisome biogenesis are similar among different organisms but not identical. Table 3 describes the full list of PEX genes so far identified and their distribution among different species as well as some characteristics of the peroxins encoded by the different PEX genes. So far, 16 different PEX genes have been identified in humans. These include HsPEX1, HsPEX2, HsPEX3, HsPEX5, HsPEX6, HsPEX7, HsPEX10, HsPEX11a, HsPEX11b, HsPEX11g, HsPEX12, HsPEX13, HsPEX14, HsPEX16, HsPEX19, and HsPEX26. We will proceed by describing the proteins encoded by these PEX genes and their presumed role in peroxisome biogenesis. Conceptually, the process of peroxisome biogenesis can be subdivided into distinct steps including (i) peroxisome membrane assembly, (ii) import of matrix proteins, and (iii) peroxisome proliferation and maintenance. In the next paragraphs, we will describe what is known about these different steps with particular emphasis on the situation in humans. We will begin by describing peroxisome membrane biogenesis and the roles of Pex3p, Pex16p, and Pex19p. Peroxisome membrane biogenesis and the human peroxins HsPEX3p, HsPEX16p, and HsPEX19p The first clue that the mechanism involved in peroxisome membrane biogenesis is fundamentally different from the one used to transport peroxisomal matrix proteins across the peroxisomal membrane was the discovery by Santos et al. (30) that cells from Zellweger patients contain peroxisome membrane structures, called ghosts, which contain PMPs but lack most, if not all, of their matrix protein content. Like the peroxisomal matrix proteins, PMPs are synthesized on free polyribosomes and imported into peroxisomes by a direct cytosol-to-peroxisome mechanism. In general, PMPs lack functional PTS1 and PTS2 signals and their import is independent of the PTS1- and PTS2-protein import routes. This is true for all bona fide integral PMPs (ipmps), whereas peripheral PMPs, like dihydroxyacetonephosphate acyltransferase (DHAPAT) and alkyl- DHAP synthase, use the PTS1- and PTS2-protein import routes. Multiple studies have attempted to define the targeting signals in ipmps. These studies have clearly shown that ipmps are not targeted to peroxisomes via carboxy-terminal or amino-terminal extensions as in PTS1 and PTS2 proteins. All data show that the targeting information is actually contained within the polypeptide chain itself. Although knowledge about targeting signals in ipmps has remained limited so far, one signal has been identified in both single- and multi-span transmembrane proteins, which is made up of a basic cluster of amino acids oriented towards the peroxisomal matrix, in front of a transmembrane span which directly follows the basic amino acid cluster. Some proteins may require additional targeting information on the cytosolic side of the peroxisomal membrane as in ScPex15p (31). There is increasing evidence, however, which suggests that ipmps are directed to peroxisomes via multiple, distinct targeting signals rather than a single targeting signal. Indeed, 112

7 Peroxisomal disorders Table 3. Overview of the different PEX genes and characteristics of their protein products (peroxins) Identified in Gene Hs Sc Yl Nc Ce Human gene locus Peroxin characteristics Subcellular localization Interacting peroxins References PEX1 þ þ þ þ þ 7q21 q22 AAA protein required for peroxisomal matrix protein import; interacts with Pex6p PEX2 þ þ þ þ þ 8q21.1 RING zinc finger protein involved in matrix protein import downstream of receptor docking Mainly cytosolic, partly peroxisomal Pex6p (21, 117, 140) Integral PMP Pex10p (141) PEX3 þ þ þ 6q23 q24 PMP import; possible docking factor for Pex19p Integral PMP Pex19p (23, 46) PEX4 þ þ E2 ubiquitin conjugating enzyme required Peripheral PMP Pex22p (24, 142, 143) for peroxisomal matrix protein import PEX5 þ þ þ þ þ 12p13.3 TPR protein; receptor for PTS1 proteins Mainly cytosolic, partly peroxisomal PEX6 þ þ þ þ þ 6p21.1 AAA protein; interacts with Pex1p, ScPex15p, and HsPex26p; required for matrix protein import Mainly cytosolic, partly peroxisomal PEX7 þ þ þ 6q21 q22.2 WD protein; receptor for PTS2 proteins Mainly cytosolic, partly peroxisomal PEX8 þ þ þ Involved in matrix protein import downstream of receptor docking PEX9 þ Involved in matrix protein import; only identified in Y. lipolytica PEX10 þ þ þ þ 1p36.32 RING zinc finger protein; required for matrix protein import; acting downstream of receptor docking PEX11 þ þ þ þ 15q25.2 (a) 1q21.1(b) 19p13.3 (g) Involved in peroxisome division and proliferation and/or transport of medium chain fatty acids PEX12 þ þ þ þ 17q21.1 RING zinc finger protein, required for matrix protein import, acting downstream of receptor docking PEX13 þ þ þ þ 2p14 p16 SH3 protein; matrix protein import; involved in receptor docking with Pex14p Pex7p, 8p, 10p, 12p, 13p, 14p Pex1p, (Sc)Pex15p, (Hs)Pex26p Pex5pL, 13p, 14p, 18p, 20p, 21p (49, 50, 55) (84, 95) (59, 62, 63, 64) Luminal PMP Pex5p, Pex20p (144, 145) Integral PMP (146) Integral PMP Pex2p, 5p, 12p, and 19p (147, 148) Integral PMP Pex19p (101, 102, ) Integral PMP Pex5p, 10p, and 19p Integral PMP Pex5p, 7p, 14p, and 19p PEX14 þ þ þ þ 1p36.22 Initial site of receptor docking PMP Pex5p, 7p, 13p, (153) 17p, and 19p PEX15 þ Required for matrix protein import; membrane anchor Integral PMP Pex6p (31, 95) for Pex6p;yeast equivalent of human Pex26p PEX16 þ þ þ 11p11.11 Required for PMP import, together with Integral PMP Pex19p (47, 154) Pex3p and Pex19p (149) (150, 151, 152) PEX17 þ Required for matrix protein import Peripheral PMP Pex14p, Pex19p (42, 79, 80) PEX18 þ Required for PTS2 protein import in S. cerevisiae; binds to ScPex7p Mainly cytosolic, partially peroxisomal PEX19 þ þ þ þ þ 1q22 Cytosolic PMP receptor Mainly cytosolic, partly peroxisomal Pex7p (27) Pex3p, 10p, 12p, 13p,14p, 16p, 17p, 11ap, 11bp (43, 155, 156) 113

8 Wanders and Waterham Table 3. (continued) Identified in Gene Hs Sc Yl Nc Ce Human gene locus Peroxin characteristics Subcellular localization Interacting peroxins References PEX20 þ þ Required for PTS2 protein import and thiolase oligomerization in Y. lipolytica PEX21 þ Required for PTS2 protein import in S. cerevisiae; binds to Pex7p PEX22 þ PMP involved in matrix protein import; membrane anchor for Pex4p Mainly cytosolic, partly peroxisomal (28) Mainly cytosolic Pex7p, 13p, 14p (27) Integral PMP Pex4p (157) PEX23 þ þ PMP involved in matrix protein import Integral PMP (158) PEX24 þ Involved in peroxisome assembly; high sequence Integral PMP (160) similarity to YlPex28p and YlPex29p PEX25 þ Involved in regulating peroxisome number, size, and Peripheral PMP Pex27p (159, 161) distribution together with Pex28p, Pex29p, and Vps1p PEX26 þ 22q11.21 Matrix protein import; recruits Pex1p Pex6p complex to Integral PMP Pex6p (96) the peroxisomal membrane PEX27 þ Controls peroxisome size and number; extensive sequence Peripheral PMP Pex25p (159, 162) similarity to Pex11p and Pex25p PEX28 þ Involved in regulating peroxisome number, size, and Integral PMP (163) distribution together with Pex25p, Pex29p, and Vps1p PEX29 þ Involved in regulating peroxisome number, size, and Integral PMP (163) distribution together with Pex25p, Pex28p, and Vps1p PEX30 þ Involved in the control of peroxisome size and proliferation, together with Pex28p, 29p, 31p, and 32p PEX31 þ Involved in the control of peroxisome size and proliferation, together with Pex28p, 29p, 30p, and 32p PEX32 þ Involved in the control of peroxisome size and proliferation, together with Pex28p, 29p, 30p, and 31p Integral PMP Pex28p, 29p, 31p, 32p Integral PMP Pex28p, 29p, 30p, 32p Integral PMP Pex28p, 29p, 30p, 31p (26) (26) (26) Abbreviations used: Hs = Homo sapiens; Sc = Saccharomyces cerevisiae; Yl = Yarrowia lipolytica; Nc = Neurospora crassa; Ce = Caenorhabditis elegans; PMP = Peripheral Membrane Proteins. 114

9 Peroxisomal disorders following earlier work by Dyer et al. (32), Wang et al. (33) reported the identification of three discrete targeting signals in S. cerevisiae PMP47. Furthermore, Jones et al. (34) showed that PMP34, the human homolog of C. boidinii PMP47, contains at least two non-overlapping sets of targeting information (amino acids and ), either of which is sufficient for insertion into the membrane. This is in contrast to data by Honsho et al. (35) who reported that PMP47 was targeted to peroxisomes via a different PTS located in the region containing amino acids Furthermore, Jones et al. (34) also identified two independent sets of targeting information in human Pex13p. In addition, Brosius et al. (36) identified two distinct, non-overlapping peroxisomal membrane-targeting signals in rat and human PMP22, one in the amino-terminal and the other in the carboxy-terminal end of the protein. Taken together, these results challenge the assumption that PMPs are targeted to peroxisomes via single PTSs and rather suggest the involvement of multiple, nonoverlapping targeting regions in ipmps. Studies in different yeast mutants as well as in fibroblasts from PBD patients have shown that ghosts are absent in some mutants indicating that in these mutants, the targeting of both peroxisomal matrix proteins and PMPs is deficient. In S. cerevisiae, the pex3 and pex19 mutants turned out to lack ghost-like structures. The same was found for human fibroblasts mutated in either the PEX3 or PEX19 gene. Furthermore, ghosts were also lacking in fibroblasts from patients mutated in PEX16. Taken together, these results indicate that Pex3p, Pex16p, and Pex19p play an essential role in peroxisome membrane biogenesis as described below. PEX3 The PEX3 gene, first cloned in S. cerevisiae by Hohfeld et al. (23) encodes a kDa protein, firmly anchored in the peroxisomal membrane with its C-terminus exposed to the cytosol, whereas opinions differ with respect to the N-terminus being either cytosolic or intraperoxisomal. The human gene was cloned in 1998 by Kammerer et al. (37). Pex3p interacts with Pex19p via its C-terminal domain. In human cells with defective PEX3, the peroxin Pex14p is mislocalized to mitochondria, whereas the peroxisomal transporters adreno leuko dystrophy protein (ALDP) and peroxisomal membrane protein of 70 kda (PMP70) are absent and less abundant, respectively. In CHO pex3d cells, Pex12p and Pex13p are absent and Pex14p less abundant. PEX19 Pex19p is a farnesylated protein first identified by James et al. (38) in CHO cells. Subsequent studies have led to the identification of a number of yeast homologs as well as human PEX19 (39). The protein is hydrophilic and contains a CAAX box allowing farnesylation of the cysteine. The exact role of Pex19p farnesylation is not resolved yet, although it may assist in peroxisomal membrane association. Indeed, in S. cerevisiae, farnesylation appears to be essential for its function (40), but this is not true for P. pastoris (15) and in humans (41). Pex19p is predominantly cytosolic, with only a small amount bound to the peroxisomal membrane, and interacts with a large variety of PMPs including peroxins: (i) Pex3p, Pex10p, Pex12p, Pex13p, Pex14p, Pex16p, and Pex17p; (ii) proteins involved in peroxisome proliferation (Pex11a and Pex11b); (iii) metabolite transporters (PMP34, PMP70, ALDP, and adreno leuko dystrophy related protein (ALDR); and (iv) PMPs of unknown function (PMP22 and PMP24) (15, 40 44). Based on these results, it is suggested that Pex19p may function as a cytosolic PMP receptor analogous to Pex5p and Pex7p, which are the cytosolic receptors for PTS1 and PTS2 proteins, respectively. Elegant experiments by Sacksteder et al. (41) in which Pex19p was directed to the nucleus by fusing it to a nuclear localization signal have provided convincing evidence in favor of this suggestion although this view is disputed by others (43, 45, 46). PEX16 In contrast to Pex3p and Pex19p, which are present in multiple mammalian and yeast species, Pex16p is lacking in most species and has only been reported in humans and the yeast Y. lipolytica (47) in which Pex16p has different properties as compared to human Pex16p playingnoroleinmembraneassembly.thehuman PEX16 gene was identified by Honsho et al. (48) and encodes a 38.6-kDa integral membrane protein with two putative membrane-spanning domains and both the N- and C-termini exposed to the cytosol. Its function is unknown. Cells defective in PEX16 lack ghosts as assessed by immunofluorescence microscopy analysis of PMP70 (48) and a range of other PMPs (12). Import of peroxisomal matrix proteins Recognition of PTS1 and PTS2 proteins in the cytosol by the import receptors Pex5p and Pex7p The realization that peroxisomal proteins are synthesized on cytosolic polyribosomes and 115

10 Wanders and Waterham contain specific targeting signals, which direct them to peroxisomes, implied the existence of receptors, recognizing the PTS1 and PTS2 sequences. The PTS1 receptor (Pex5p) was first identified in 1993 in the yeasts P. pastoris (49) and S. cerevisiae (50). Subsequent studies have led to the identification of orthologs of Pex5p in a range of different species including humans (51 53). Pex5p binds PTS1 proteins in the cytosol and cycles between the cytosol and the peroxisome. In most organisms, Pex5p is mainly localized in the cytosol with only a small fraction being associated with peroxisomes. Based on these data, a model has been proposed called the shuttlemodel in which Pex5p binds its cargo, i.e. a PTS protein, in the cytosol, after which the receptor cargo complex docks at the peroxisomal membrane followed by dissociation of the complex and transport of the PTS1 protein across the membrane and recycling of the receptor back into the cytosol (Fig. 2a). In the yeast Y. lipolytica, however, Pex5p is mainly intraperoxisomal (54). Similar observations have been made in other yeasts including H. polymorpha (55). This dual localization of Pex5p has led to a revised model for protein import into peroxisomes in which the Pex5p PTS1 protein complex is translocated across the peroxisomal membrane in toto followed by recycling of the receptor back into the cytosol. Recent work by Dammai and Subramani (56) suggests that such a so-called extended shuttle model may also apply to the human situation (Fig. 2b). Pex5p belongs to the family of TPR-containing proteins, which are characterized by highly degenerate, repetitive sequences of 34 amino acids. TPRs are found as tandem arrays of 3 16 motifs in a wide variety of proteins involved in many different cellular processes including cell-cycle regulation, chaperone functions, and protein phosphorylation. The C-terminal half of Pex5p consists of two clusters each comprising three TPR domains (TPR 1 3 and TPR 5 7), which are linked by a hinge region denoted TPR4. The TPR domains participate in a special folding structure that allows the interaction with the PTS1 tripeptide that appears to be embraced by all TPR motifs (57, 58). The importance of the TPR domains for recognition of the PTS1 tripeptide is immediately clear if it is realized that a single amino acid change (N489K) within the sixth TPR domain abolishes interaction between human Pex5p and the PTS1 signal and causes NALD, one of the Zellweger spectrum disorders (51). In addition to binding PTS1 proteins, all Pex5p proteins bind Pex13p and Pex14p whereas mammalian Pex5p proteins also bind Pex7p as discussed later. Pex7p, the receptor for PTS2 proteins. The identification by Erdmann et al. (20) of an S. cerevisiae mutant with a defect in PTS2-mediated import, but a normal PTS1-import pathway, led Kunau and coworkers to identify the PTS2 receptor (Pex7p) (59) which turned out to be a member of the WD-40 family of proteins, a family characterized by repeats of approximately 40 amino acid residues, each containing a central Trp-Asp (WD) motif. WD-40 proteins have been implicated in interactions with TPR-containing proteins and recent evidence suggests that Pex5p and Pex7p indeed interact, at least in mammals as discussed below. After its initial identification in S. cerevisiae Original shuttle model Extended shuttle model Receptor Cytoplasm Peroxisomal membrane Peroxisomal matrix 116 Peroxisomal matrix protein Receptor Cytoplasm Peroxisomal membrane Peroxisomal matrix Fig. 2. Schematic representation of the original and modified shuttle models. (a) The original shuttle model in which the receptor shuttles between the cytosol, where a PTS1 or PTS2 protein is picked up, and the cytosolic face of the peroxisomal membrane, where the receptor cargo complex docks followed by dissociation of the receptor cargo complex and transfer of the cargo protein across the peroxisomal membrane and recycling of the receptor back into the cytosol. (b) The modified so-called extended shuttle model in which the receptor cargo crosses the peroxisomal membrane en block followed by back transport of the empty receptor from the inside of peroxisomes to the cytosol.

11 Peroxisomal disorders (59 61), subsequent Pex7p proteins have been identified in other species including mammals (62 64). The subcellular localization of Pex7p is still controversial due to conflicting results in human fibroblasts and S. cerevisiae, which show a predominant cytosolic localization in human fibroblasts vs an entirely peroxisomal localization in S. cerevisiae as concluded by Zhang and Lazarow (60). PTS2 protein import route in mammals and yeasts: similar game, different players (HsPex5pL, ScPex18p/Pex21p, and YlPex20p/ NcPex20p). Despite the many similarities between mammals and yeasts with respect to peroxisome biogenesis, there are also important differences, one being the role of Pex5p. Indeed, in yeasts, Pex5p is only involved in the import of PTS1 proteins, whereas in mammals, Pex5p is involved in both PTS1- and PTS2-protein import. In contrast, Pex7p is involved in PTS2-protein import only, whichistrueforbothmammalsandyeasts.the exclusive role of Pex5p and Pex7p in PTS1- and PTS2-protein import, respectively, in yeasts, is exemplified by the phenotypes of the pex5 and pex7 yeast mutants in which only the import of PTS1 proteins (pex5-mutant) or PTS2 proteins (pex7-mutant) is impaired. Subsequent studies revealed that in human skin fibroblasts and CHO cells, the situation is different with respect to Pex5p. In fact, analysis of human and CHO pex5-mutants revealed two different phenotypes; in some mutants, only the PTS1-protein import pathway was disrupted whereas in other mutants, both the PTS1- and PTS2-protein import pathways were blocked. This enigma was resolved when Pex5p was found to exist in two forms in mammals: a long form (Pex5pL) and a short form (Pex5pS). Pex5pS and Pex5pL are identical with one important difference, which is the presence of an additional internal segment of 37 amino acids positioned between amino acids 214 and 215 (human) or 215 and 216 (Chinese hamster). The differential role of Pex5pS and Pex5pL was clearly shown by Otera and coworkers (65). These authors showed that in pex5-deficient CHO cells disturbed in both PTS1- and PTS2-protein import, Pex5pS only restored the import of PTS1-proteins whereas Pex5pL restored both PTS1- and PTS2-import in the same cells. The same was shown by Braverman et al. (66) in human pex5-mutants in which Pex5pL restored both PTS1- and PTS2-protein import whereas Pex5pS restored PTS1-protein import only. These data clearly show that Pex5pL plays an essential role in PTS2-protein import. Subsequent studies have shown that Pex5pL and Pex7p interact with one another. The region in Pex5pL necessary for this interaction was mapped using truncated versions of Pex5pL and includes the amino-terminal amino acids of the Pex5pL-specific 37 amino acids insertion, together with amino acids lying outside this region. The S214F mutation in this region disrupted binding to Pex7p as shown by Otera and coworkers (67) and resulted in a specific PTS2-protein import defect, while PTS1- protein import was not affected (68). It is important to stress that the role of Pex5pL in the import of PTS2 proteins in mammals is independent of its role in the import of PTS1 proteins. Indeed, when a truncated version of Pex5pL was expressed containing only the amino-terminal half of the protein without a TPR motif, complementation of the PTS2-protein import defect was observed in PEX5- deficient mammalian cells (67, 69). Recent studies have shed new light on the remarkable difference with respect to the role of Pex5p between yeasts and mammals. Studies in the yeast S. cerevisiae have shown that PTS2-protein import is not only dependent on Pex7p but also on Pex18p and Pex21p (27), which are not found in humans. In Y. lipolytica (28) and N. crassa (29), PTS2-protein import is dependent upon another protein named Pex20p. It turns out that the amino acid sequence in the 37 amino acid internal region of Pex5pL is highly conserved in S. cerevisiae Pex18p and Pex21p and Y. lipolytica and N. crassa Pex20p, suggesting that this region, shared between the four proteins, is involved in the formation of an import-competent complex of Pex7p and PTS2 proteins. The absence of the conserved peptide motif in fungi is in line with the fact that Pex5p plays no role in PTS2- protein import, whereas its presence in different mammals, protozoa, and plants indicates that Pex5p is also involved in PTS2-protein import in these organisms. Figure 3 depicts the different PTS2-protein import pathways and the distinct roles of HsPex5pL, ScPex18p and ScPex21p, and YlPex20p/NcPex20p in the different species. Interestingly, Pex5p of Caenorhabditis elegans does not contain this conserved peptide motif, which agrees with the complete lack of the PTS2-protein import pathway in this organism (70). Receptor docking and the essential role of human Pex13p and Pex14p There is general agreement that Pex13p and Pex14p form the docking complex where the PTS1- and PTS2-protein import routes converge. Pex13p is a PMP with two membrane-spanning domains with both the N- and C-terminus exposed to the cytosol. Pex13p belongs to the family of SH3 (Src-Homology 3) proteins. The SH3 domain in Pex13p is located at its C-terminus. SH3 domains are small, non-catalytic 117

12 Wanders and Waterham PTS2p + Pex7p Pex 5pL Pex20p PTS2p Pex7p H. sapiens S. cerevisiae Y. lipolytica N. crassa Pex18p Pex21p PTS2p Pex7p Pex 5pL PTS2p Pex7p Pex20p Pex18p PTS2p Pex7p Pex21p Pex 14p Pex 13p Fig. 3. PTS2-protein import in mammals and yeast: similar game, different players. In several organisms, including humans (Homo sapiens) the long form of the PTS1 receptor (Pex5pL) is needed for the import of PTS2 proteins by forming a complex with Pex7p and its cargo, i.e. a PTS2 protein. In Saccharomyces cerevisiae, PTS2-protein import requires the active participation of two helper proteins (Pex18p and Pex21P) whereas in Yarrowia lipolytica and Neurospora crassa, this task is fulfilled by a single protein, i.e. Pex20p. protein modules capable of protein protein interactions, which participate in diverse intracellular processes. These domains consist of amino acids, have a high sequence similarity, and form structurally similar conformations. Resolution of the three-dimensional structure of various SH3 domains and their contact sites with peptide ligands has revealed that highly conserved aromatic amino acid residues form a hydrophobic cleft running between two variable loops: This hydrophobic cleft forms the binding platform for ligand association, with the two variable loops contributing to ligand recognition and specificity. Typically, SHR domains recognize and bind short proline-rich peptides. The minimal consensus sequence for this peptide is Pro-X-X-Pro (P-X-X-P), where X is any amino acid, plus an additional basic amino acid located C-terminally (class I: P-X-X-P-X-R) or N-terminally (class II: R-X-X-P-X-X-P) of the P-X-X-P core. The proline-rich peptide segment adopts a left-handed polyproline-type helix (PP2) that, depending on the class of the ligand, canbindintwoorientationswithrespecttothe SH3 domain. Pex14p is the other member of the docking machinery and is required for the import of both PTS1 and PTS2 proteins. Pex14p is tightly associated with the peroxisomal membrane either as a peripheral membrane protein or an integral membrane protein. Pex14p interacts with Pex13p but can also bind directly to Pex5p and Pex7p. Schliebs et al. (71) have shown that the aminoterminal 78 amino acids of human Pex14p are involved in binding of Pex5p with a very high affinity. Multiple binding sites for Pex14p were shown to be present in the amino-terminus of Pex5p, and subsequent studies showed that the pentapeptide WXXXF/Y repeats were involved in binding Pex14p in mammals and in plants 118

13 Peroxisomal disorders (67, 72, 73). The number of pentapeptide repeats differs among the different organisms with two repeats in S. cerevisiae Pex5p, and seven in human Pex5pL. Pex14p directly interacts with Pex13p via the SH3 domain which involves a class II P-X-X-P-X-R motif (PPTLPHRDW) in Pex14p as shown for S. cerevisiae (74). Binding of Pex5p to the same SH3 domain of Pex13p does not occur at the PP-2-binding phase but at a novel interaction site (75). X-ray crystallography and mass spectrometry data from Dounagamath and coworkers (76, 77) revealed the existence of two functionally and structurally independent binding sites on the SH3 domain of Pex13p for Pex5p and Pex14p, respectively, with Pex7p binding at the amino-terminal end of the Pex13p. Although it was initially thought that Pex13p was the first site of receptor docking, current data suggest that it is in fact Pex14p, which comes first. This model is supported by data from Otera et al. (67) and Urquhart et al. (78) who showed that Pex14p binds to PTS1-loaded Pex5p whereas Pex13p only binds to unloaded Pex5p. Otera et al. (67) proposed that Pex5p bound to a PTS1 protein first binds to the Pex13p Pex14p complex via interaction with Pex14p after which the PTS1 protein is released from Pex5p followed by dissociation of the Pex13p Pex14p complex. Subsequently, the unloaded Pex5p is transferred to Pex13p and shuttles back to the cytosol. This model implies that Pex13p and Pex14p form functionally distinct subcomplexes, which are both involved in the import process of peroxisomal proteins. Taken all data together, Pex14p is indeed the most likely primary docking protein. It might well be that other proteins are part of the docking complex. A good candidate, identified in S. cerevisiae (79) and P. pastoris (42), is Pex17p which behaves as a peripheral membrane protein tightly bound to the peroxisomal membrane in S. cerevisiae (79) whereas in P. pastoris, it is an integral membrane protein with the carboxyterminus facing the cytosol (42). In S. cerevisiae, Pex17p is thought to be part of the docking complex together with Pex14p and Pex13p (79), whereas Snyder et al. (42) favored a model in which Pex17p is also involved in the import of PMPs. Subsequent studies by Hettema et al. (25) and Harper et al. (80) showed normal import of PMPs in both Ppex17pD and Scpex17D cells, which argues against the model of Snyder et al. (42). Taken together, the bulk of evidence favors a role of Pex17p in peroxisomal matrix importandnotintheimportofpmps.except from its identification in S. cerevisiae and P. pastoris, no mammalian Pex17p has been identified so far. Translocation across the peroxisomal membrane and the human peroxins Pex2p, Pex10p, and Pex12p Three peroxins belonging to the family of RING zinc finger proteins, i.e. Pex2p, Pex10p, and Pex12p, are thought to be involved in the actual transport machinery. All three proteins are ipmps and have a carboxy-terminal RING finger domain exposed to the cytosol. Based on the finding that fibroblasts from PBD patients with mutations in PEX2, PEX10, or PEX12 accumulated Pex5p at the level of peroxisomes in contrast to normal fibroblasts, Pex2p, Pex10p, and Pex12p are thought not to be involved in receptor docking but in one of the subsequent steps of protein import. Mutant pex2, pex10,orpex12 cells are all disturbed in the import of peroxisomal matrix proteins while the import of PMPs is not affected. Reguenga et al. (81) have obtained evidence suggesting that Pex2p and Pex12p are together in a complex with Pex14p and Pex5p (81). Pex13p is also part of this complex although in nonstoichiometric amounts. Another prediction for the proteins involved in translocation would be that they should interact directly or indirectly either with the cargo to be translocated or with the receptors for that cargo, Pex5p and/or Pex7p. Pex2p, Pex10p, and Pex12p all contain a C3HC4 zinc-binding domain, or RING finger, a protein module that is thought to mediate protein protein interactions. The RING finger is essential for the functions of both Pex10p and Pex12p, and recent studies have shown that Pex10p and Pex12p directly interact with Pex5p and with each other (82, 83). Receptor recycling and the role of the human peroxins Pex1p, Pex6p, and Pex26p The peroxins Pex1p and Pex6p are members of the large family of AAA proteins (ATPases) associated with a wide range of cellular activities (21, 84). The AAA domain consists of amino acids and contains two motifs named Walker A and B, which bind and hydrolyze ATP, respectively (85). The role of Pex1p and Pex6p in peroxisome biogenesis has remained controversial with two opposing views. The first view holds that Pex1p and Pex6p are required for peroxisome biogenesis possibly playing a role in some membrane fusion event involving vesicles derived from the ER (86). This hypothesis was stimulated, in part, by the observation that many of the initially identified members of the AAA ATPase family were involved in membrane fusion events. In line with this postulate, Titorenko et al. (87) showed that in Y. lipolytica, Pex1p and 119