REVIEW ARTICLE Biogenesis of peroxisomes Topogenesis of the peroxisomal membrane and matrix proteins Ines Heiland and Ralf Erdmann Ruhr-Universitat Bochum, Institut fur Physiologische Chemie, Bochum, Germany ă ă Keywords peroxin, peroxisome, protein transport Correspondence R Erdmann, Ruhr-Universitat Bochum, ă Institut fur Physiologische Chemie, ă Abteilung fur Systembiochemie, ă 44780 Bochum, Germany Fax: +49 234 321 4266 Tel: +49 234 322 4943 E-mail: ralf.erdmann@rub.de (Received 10 February 2005, accepted 31 March 2005) doi:10.1111/j.1742-4658.2005.04690.x Genetic and proteomic approaches have led to the identification of 32 proteins, collectively called peroxins, which are required for the biogenesis of peroxisomes Some are responsible for the division and inheritance of peroxisomes; however, most peroxins have been implicated in the topogenesis of peroxisomal proteins Peroxisomal membrane and matrix proteins are synthesized on free ribosomes in the cytosol and are imported post-translationally into pre-existing organelles (Lazarow PB & Fujiki Y (1985) Annu Rev Cell Biol 1, 489–530 [1]) Progress has been made in the elucidation of how these proteins are targeted to the organelle In addition, the understanding of the composition of the peroxisomal import apparatus and the order of events taking place during the cascade of peroxisomal protein import has increased significantly However, our knowledge on the basic principles of peroxisomal membrane protein insertion or translocation of peroxisomal matrix proteins across the peroxisomal membrane is rather limited The latter is of particular interest as the peroxisomal import machinery accommodates folded, even oligomeric, proteins, which distinguishes this apparatus from the well characterized translocons of other organelles Furthermore, the origin of the peroxisomal membrane is still enigmatic Recent observations suggest the existence of two classes of peroxisomal membrane proteins Newly synthesized class I proteins are directly targeted to and inserted into the peroxisomal membrane, while class II proteins reach their final destination via the endoplasmic reticulum or a subcompartment thereof, which would be in accord with the idea that the peroxisomal membrane might be derived from the endoplasmic reticulum Introduction Peroxisomes are ubiquitious, single membrane bound organelles of eukaryotic cells [2] They maintain various functions that differ depending on the species and cell type, as well as the environmental or developmental conditions Many metabolic pathways of peroxisomes lead to the production of hydrogen peroxide The subsequent decomposition of this toxic compound by catalase is a fundamental process that takes place in almost all peroxisomes Moreover, peroxisomes contribute to the b- and a-oxidation of fatty acids, synthesis of ether lipids such as plasmalogens, and the oxidation of bile acids and cholesterol [3–6] Defects in the biogenesis of peroxisomes are the molecular cause for severe inherited diseases, called peroxisome biogenesis disorders Abbreviations APX, ascorbate peroxidase; mPTS, membrane protein targeting signals; PMP, peroxisomal membrane protein; PTS, peroxisomal targeting signal; TPR, tetratricopeptide repeat 2362 FEBS Journal 272 (2005) 2362–2372 ª 2005 FEBS I Heiland and R Erdmann (PBD,) such as Zellweger syndrome, neonatal adrenoleukodystrophy and Refsums disease [7] Peroxisomal matrix protein import Many investigations have focussed on the elucidation of the import of peroxisomal matrix proteins, and the mechanisms involved are becoming better understood [8,9] It is generally accepted that Pex5p and Pex7p, the receptors for the proteins harboring peroxisomal targeting sequences, cycle between the cytosol and the peroxisome This gave rise to the so-called model of shuttling receptors [10,11] According to this model, the import receptors bind cargo proteins in the cytosol and direct them to a docking and translocation complex at the peroxisomal membrane There, the cargo is released and translocated across the peroxisomal membrane while the receptor shuttles back to the cytosol in a so-far unknown manner The so-called extended shuttle hypothesis is based on the assumption that the import receptor does not stop at the peroxisomal membrane but enters the peroxisomal lumen together with its cargo [12–14] In this case, cargo release takes place in the peroxisomal matrix and the cargounloaded receptors are transported back to the cytosol Peroxisomal targeting sequences and their receptors Peroxisomal matrix proteins are synthesized on free ribosomes in the cytosol and are bound by the peroxisomal targeting sequence receptors Pex5p and Pex7p To date, two targeting sequences for peroxisomal matrix proteins have been identified The most abundant is the peroxisomal targeting signal type I (PTS1), which consists of a conserved tripeptide at the extreme C-terminus of the protein and a less conserved upstream region [15,16] The consensus sequence of the C-terminal tripeptide is S ⁄ A-K ⁄ R-L ⁄ M, but not all variations are functional in all species [17–20] The second peroxisomal targeting signal (PTS2) is located close to the N-terminus and is defined by the less conserved consensus sequence R-L ⁄ I-X5HL [20,21] The PTS1 receptor Pex5p contains seven tetratricopeptide repeat (TPR) domains, which are essential for PTS1 binding [22] Of these seven TPR domains six interact directly with the tripeptide, whereas TPR4 is important for the structural alignment of the other TPR motifs [23,24] Acyl-CoA oxidases from Saccharomyces cerevisiae, Hansenula polymorpha and Candida tropicalis contain neither a PTS1 nor a PTS2 signal However, it has been shown that these proteins are still targeted via the PTS1 receptor Pex5p, but bind to FEBS Journal 272 (2005) 2362–2372 ª 2005 FEBS Biogenesis of peroxisomes regions of the protein distinct from the PTS1-recognition domain [25] Pex7p is the cytosolic receptor for PTS2 proteins and belongs to the family of WD40 proteins that share a consensus sequence of 40 amino acids, which contains a central tryptophan-aspartic acid motif [10] Pex7p contains six of these repeats In S cerevisiae, Pex7p is associated with Pex18p ⁄ Pex21p [26,27], proteins with redundant functions that are presumed to mediate the association of cargo-loaded Pex7p with the docking complex Whereas Pex7p is present in nearly all species analysed, Pex18p and Pex21p are evolutionarily less conserved In Neurospora crassa and Yarrowia lipolytica the function of Pex18p ⁄ Pex21p is performed by Pex20p, suggesting that the protein is a true orthologue of the yeast proteins [28,29] In addition to the fact that PTS1 and PTS2 protein import pathways employ different components there seems to be a common mechanism for both processes In support of this assumption, it has been shown that Pex18p can functionally replace the N-terminal domain of Pex5p [30] Remarkably, in humans, Pex5p exists in two isoforms, one characterized by a 37 amino acid insertion that mediates binding of Pex7p to Pex5p and therefore overcoming the requirement for Pex18p ⁄ Pex21p [31,32] Thus, in mammalian cells, the PTS2 pathway depends on the presence of the long isoform of PTS1 receptor Pex5p, which is required to direct cargo-loaded Pex7p to the import machinery at the peroxisomal membrane [29–32] Furthermore, it has been demonstrated recently that PTS1 and PTS2 import pathways are also coupled in plants [33] The peroxisomal protein import machinery Upon the binding of PTS1 proteins, Pex5p depolymerizes [34] and is transported to the peroxisome where it interacts with Pex14p [35–39] and Pex13p [40–44], as well as Pex12p [45–48], leading to the question of which of these proteins performs the docking event As Pex5p accumulates at the peroxisomal membrane in pex13-, pex2- and pex12- but not in pex14-deficient cell lines [49] and as the binding affinity of cargo-loaded Pex5p is much higher for Pex14p then for Pex13p [50,51], Pex14p is believed to mediate peroxisomal membrane association of Pex5p At the peroxisomal membrane, Pex14p is associated with Pex17p [52] and at least temporally with Pex13p The puative peroxisomal import complex (importomer) is formed by the RING-finger subcomplex containing Pex2p, Pex10p and Pex12p, and the docking complex comprising 2363 Biogenesis of peroxisomes Pex13p, Pex14p and Pex17p Both subcomplexes are linked via Pex8p [53], which contains both targeting sequences for peroxisomal matrix protein import (PTS1 and PTS2) However, the import of Pex8p does not depend on these signals [54,55] It is imaginable that these targeting signals are bound by the import receptors after cargo release to prevent reassociation with cargo proteins and evidence has been provided for Pex8p being directly involved in cargo– receptor dissociation [56] The functions of other components of the import complex are still unknown Whether the RING-finger complex is really involved in peroxisomal matrix protein import or rather in the re-export of the PTS1 receptor Pex5p still has to be investigated It has been demonstrated that Pex5p becomes ubiquitinated during import [57–59] Furthermore, Pex18p, a component of the signal recognition complex in the PTS2-pathway, becomes mono- and diubiquitinated during import and is degraded in a proteasome-dependent manner [60] Polyubiquitination of Pex5p is detectable in pex1, pex6, pex4 and pex22 mutants of S cerevisiae and requires a functional import complex The physiological relevance of Pex5p ubiquitination, however, remains to be shown It is possible that import receptors that remained in the import pathway are polyubiquitinated and subsequently directed to proteasomal degradation as a form of quality control [58] However, it is also conceivable that ubiquitination of Pex5p and Pex18p serves as a signal for their export back to the cytosol [57,59] As RING-finger proteins often function as E3–ubiquitin protein ligases in ubiquitin and ubiquitin-like conjugations [61], Pex2p, Pex10p and Pex12p might be involved in the ubiquitination of the import receptor Pex5p recycling to the cytosol has been demonstrated to be accompanied by ATP hydrolysis and to require the N-terminus of the receptor [62,63] The current understanding of the organization of the peroxisomal import machinery for PTS1 proteins is summarized in Fig In the absence of cargo protein, Pex5p is retained in the cytosol in a tetrameric complex Upon PTS1–protein binding, Pex5p disaggregates into dimers [34] and is transported in a currently unknown manner to the peroxisome At the peroxisomal membrane, Pex5p binds to the docking complex, presumably mediated by Pex14p How the cargo or the cargo–receptor complex is translocated across the peroxisomal membrane is completely unknown Elucidation of this cellular process is a particular challenge, as the proteins are transported in a folded or even oligomeric conformation Pex8p triggers the association of the docking and the RING-finger complex 2364 I Heiland and R Erdmann and might contribute to cargo release At the end of the pathway, Pex5p is recycled back to the cytosol in an ATP-dependent manner Lipid transport to peroxisomes The major lipid components of peroxisomal membranes are phosphatidylcholine and phosphatidylethanolamine [64–66] Most enzymes involved in the synthesis of polar lipids are localized in the endoplasmic reticulum (ER), and the peroxisome is not capable of synthesizing these lipids [65,67] Therefore the lipids have to be tranported from the ER to the peroxisome, which might require the employment of specialized vesicles as postulated by Purdue and Lazarow [68] As an alternative, membrane constituents might flip from the ER membrane at contact sites between ER and peroxisomes Evidence has been provided that the latter mechanism is employed for the transport of phospholipids from the ER to mitochdondria [69–71] How peroxisomes gain their phospholipids remains to be investigated Peroxisomal membrane protein import Most mutants that are defective for the import of PTS1 and PTS2 proteins still import peroxisomal membrane proteins Thus, the import of peroxisomal membrane and matrix proteins is independent [41,42,72] The peroxisomal membrane protein targeting signals (mPTS) were identified for several peroxisomal membrane proteins (PMPs) These targeting sequences contained a basic amino acid sequence in conjunction with at least one transmembrane region [73–77] Some PMPs have been shown to posses multiple targeting signals [55,78,79] One possible reason for the existence of multiple mPTS might be that they are required to distinguish targeting to different peroxisome populations [55] This might be of particular interest for higher eukaryotes such as plants, which generate different types of peroxisomes during their development Only three of the 32 peroxins identified so far – Pex3p, Pex16p and Pex19p – have been shown to be involved in peroxisomal membrane protein import [80,81] PEX16-deficient cell lines lack detactable peroxisomal membrane structures [77,80,82] Moreover, Arabidopsis thaliana pex16 mutants show defects in oil body and fatty acid synthesis [83,84] How Pex16p participates in peroxisomal membrane biogenesis is not known The function and characteristics of Pex3p and Pex19p are discussed below FEBS Journal 272 (2005) 2362–2372 ª 2005 FEBS I Heiland and R Erdmann Biogenesis of peroxisomes Fig PTS1-import model Newly synthesized peroxisomal matrix proteins are recognized by receptors in the cytosol Upon PTS1–protein binding, the tetrameric Pex5p disaggregates into dimers and is transported to the peroxisome At the peroxisomal membrane, Pex5p binds to the docking complex comprising Pex13p, Pex14p and Pex17p How the cargo is translocated across the peroxisomal membrane is completely unknown Pex8p triggers the association of the docking and the RING-finger complex (Pex2p, Pex10p and Pex12p) and may contribute to cargo release The function of the RING-finger complex is still unknown At the end of the import cascade, Pex5p is recycled back to the cytosol in an ATP-dependent manner Pex19p – chaperone, import receptor or both? The functional role of Pex19p in peroxisome biogenesis has been controversial Pex19p is a predominantly cytosolic protein that can be farnesylated [85,86] In cells lacking Pex19p, peroxisomal membrane proteins are unstable or mislocalized [81,87] Pex19p is known to bind multiple PMPs [88], but whether it binds to the targeting signals of these proteins and therefore functions as cytosolic receptor or whether Pex19p binds unspecifically to hydrophobic regions – similar to chaperones – is still a matter of debate [89,90] However, using in vitro binding studies and bioinformatic approaches Rottensteiner et al [91] recently identified a consensus sequence for the binding sites of Pex19p These binding sites were demonstrated to be required for peroxisomal membrane protein targeting Moreover, in conjunction with an adjacent transmembrane domain, these sites proved to be sufficient for the peroxisomal membrane targeting of an otherwise mislocalized fusion protein Thus, the mPTS is formed by the Pex19p binding site together with an adjacent transmembrane segment In this assembly, the Pex19p binding site is proposed to contain the required FEBS Journal 272 (2005) 2362–2372 ª 2005 FEBS targeting information, while the transmembrane segment is required for the permanent insertion of the protein into the peroxisomal membrane The fact that the Pex19p binding site is an integral part of the mPTS also demonstrates that Pex19p functions as a targeting sequence receptor for peroxisomal membrane proteins There is, however, one exception Pex3p targeting is not dependent on Pex19p, and Pex19p binds to Pex3p in regions different from its targeting signal [90,92] Therefore, the existence of distinct classes of peroxisomal membrane proteins have been postulated [93,94] Class I PMPs are synthesized on free ribosomes in the cytosol and require Pex19p for their posttranslational import into the peroxisome Class II PMPs, such as Pex3p, are targeted to the peroxisome independent of Pex19p [92] The function of Pex19p as an mPTS receptor does not exclude that binding could contribute to the stability of the proteins [95] In fact, Pex19p has been shown to increase the half-life of newly synthesized membrane proteins in vivo [78], and it has been demonstrated to bind to in vitro synthesized Pmp22p and thereby maintain its solubility [92] This could be explained by mPTS itself being rather hydrophobic, and thus, if not shielded from hydrophobic environment, it might 2365 Biogenesis of peroxisomes contribute to misfolding and aggregation In some cases, the Pex19p binding site may even overlap with transmembrane regions of PMPs Therefore, Pex19p could indeed play a dual role in peroxisomal membrane protein import – as a general import receptor for PMPs and, probably as a consequence of mPTS binding, also as a PMP-specific chaperone Pex3p – anchor protein for Pex19p at the peroxisomal membrane Pex3p is a peroxisomal membrane protein that interacts with Pex19p at the peroxisomal membrane [96] The N-terminal region of Pex3p contains its peroxisomal targeting signal, whereas its C-terminus binds Pex19p at regions distinct from the PMP binding site The interaction of Pex19p with Pex3p is essential for peroxisomal membrane protein import, suggesting that Pex3p functions as a receptor for Pex19p at the peroxisomal membrane [92,93] It is now thought that Pex19p recognizes newly synthesized PMPs in the cytosol and directs them to the peroxisomal membrane, probably via binding to Pex3p How peroxisomal membrane proteins insert into the membrane remains to be investigated As outlined above, the topogenesis of Pex3p seems to be different from that of other PMPs The N-terminal 50 amino acids of Pex3p have been shown to be associated with vesicles that are located close to the nucleus in Dpex3 mutants of H polymorpha Furthermore, these vesicles are reported to be capable of forming mature peroxisomes after complementation with full length Pex3p [97] The first 16 amino acid of Pex3p lead to targeting of reporter constructs to the ER [98] Whether this targeting sequence is functional in the endogenous Pex3p is not known Involvement of the endoplasmic reticulum in peroxisome biogenesis In early years, it was assumed that peroxisomes originate through budding from the endoplasmic reticulum [99] In 1984, however, Fujiki and coworkers demonstrated that the peroxisomal membrane protein Pmp22p is synthesized on free ribosomes in the cytosol and imported post-translationally directly into peroxisomes [100] Based on these and other data, the ‘growth and division model’ was postulated by Lazarow and Fujiki in 1985 [1] The model postulates that all peroxisomal matrix as well as peroxisomal membrane proteins are synthesized on free ribosomes in the cytosol and are imported post-translationally into preexisting peroxisomes which then start to grow and multiply by division A major implication of this 2366 I Heiland and R Erdmann model is that peroxisomes cannot originate de novo as known for mitochondria and chloroplasts However, based on data difficult to reconcile with this model, the involvement of the ER in peroxisome biogenesis was reconsidered For example, treatment of H polymorpha cells with Brefeldin A (a fungal toxin that interferes with ER-to-Golgi transport) led to the accumulation of peroxins in ER-like structures [101] In plants treated with Brefeldin A, ascorbate peroxidase (APX) accumulates in a reticular circular network that resembles the ER but does not contain typical ER-resident proteins such as calreticulin, BiP2 and calnexin [102] In human fibroblasts, however, treatment with Brefeldin A has no effect on peroxisome biogenesis and localization of peroxisomal membrane proteins in ER-like structures has never been observed [103,104] Inactivation of the endoplasmic reticulum protein translocation factor, Sec61p, or its homologue Ssh1p from S cerevisiae, did not lead to defects in the targeting of Pex3p or peroxisome biogenesis ([105]; I Heiland & R Erdmann, unpublished data), while Titorenko and Rachubinski detected a transient colocalization of peroxins with the ER marker protein Kar2p and a cytosolic mislocalization of thiolase and alcohol oxidase in secretory pathway mutants (secmutants) of Yarrowia lipolytica [107] Furthermore, evidence for involvement of the ER in peroxisome biogenesis was provided by Mullen and coworkers, who demonstrated that tail-anchored peroxisomal membrane proteins such as APX and Pex15p are imported into plant microsomes in vitro, whereas Pmp45p is imported directly into peroxisomes [102,108] Furthermore, Tabak and coworkers reported on reticular structures observed in untreated mouse dendritic cells that contained PMPs and were connected to the smooth ER [109,110] Taken together, there is striking evidence for an involvement of the ER in peroxisome biogenesis However, the data are clear in that the standard secretion pathway is not involved The only way to reconcile these facts seems to propose the existence of a new route for the insertion of peroxisomal proteins into the ER membrane In this respect, it is interesting to note that several new routes for protein transport into the ER have been identified in recent years that not or only partially employ the standard secretion pathway One of these novel import pathways into the ER is the topogenesis of Ist2p The import of Ist2p is mRNAdependent and takes place at the cortical ER of the daughter cell [111] Whether this process requires Sec61p is unknown An example of sec-independent import into the ER is the sorting of Nyv1p This tail-anchored protein has been shown to be imported FEBS Journal 272 (2005) 2362–2372 ª 2005 FEBS I Heiland and R Erdmann Biogenesis of peroxisomes Fig Model of peroxisomal membrane biogenesis Peroxisomal class I membrane proteins are synthesized on free ribosomes in the cytosol, where they are recognized by the import receptor Pex19p that directs them to the peroxisomal membrane Membrane association of the Pex19p receptor– cargo complex is mediated by Pex3p How membrane protein insertion takes place still remains to be investigated Topogenesis of class II PMPs is independent of Pex19p Accumulating evidence suggests that PMPs class II might be targeted to the ER prior to their transport to peroxisomes Again, how these proteins reach the ER and their final destination in the peroxisomal membrane is unknown post-translationally into the ER independent of the sec-machinery [112] The mechanisms employed for tail-anchored proteins have not yet been identified It has been shown recently that the signal recognition particle can bind tail-anchored proteins, but the functional significance for the insertion process remains to be demonstrated However, in contrast to the import of secretory proteins, tail-anchored proteins are bound post-translationally by the signal recognition particle [113] Interestingly, Pex15p and APX have been shown to contain their targeting signal within their C-terminal tails [108,114] and their targeting sequences have characteristics of tail-anchored proteins [112] Moreover, APX has been shown to colocalize with tail-anchored green fluorescent protein [115] It will be interesting to investigate whether PMPs are transported into the ER via one of these novel routes or whether they employ a novel, unidentified transport pathway into the ER membrane Two distinct import pathways for PMPs? Taken together the results obtained on the import of peroxisomal membrane proteins suggest that there are at least two distinct classes of peroxisomal membrane proteins (Fig 2) The first, class I PMPs, are posttranslationally directly inserted into the peroxisomal FEBS Journal 272 (2005) 2362–2372 ª 2005 FEBS membrane in a Pex19p- and Pex3p-dependent manner The second are class II PMPs, such as Pex3p and tailanchored peroxisomal membrane proteins (e.g Pex15p and APX) that are supposed to be targeted to a thus far uncharacterized circular reticular membrane compartment, namely peroxisomal ER or peroxisomal reticulum These reticular structures may, at least temporally, be connected to the ER or may even represent an ER subdomain [110] Consequently, newly synthesized proteins of class II might first be inserted into the ER membrane before they reach their final destination in the peroxisomal membrane in an unknown fashion Nevertheless, in the presence of mature peroxisomes these proteins might also behave like PMPs of type I and thus be imported preferentially directly into peroxisomes In the absence or deficiency of peroxisomal membranes, these proteins might be imported into the reticular structures and contribute to the de novo synthesis of peroxisomes Whether the topogenesis pathway of these PMPs shares components with other sec-independent transport pathways remains to be investigated Acknowledgements We thank Hanspeter Rottensteiner and Wolfgang Schliebs for reading the manuscript Ines Heiland was supported by a Boehringer Ingelheim Fonds fellow2367 Biogenesis of peroxisomes ship This work was supported by grants from the Deutsche Forschungsgesellschaft (Er178 ⁄ 2–4) and by the Fond der Chemischen Industrie References Lazarow PB & Fujiki Y (1985) Biogenesis of peroxisomes Annu Rev Cell Biol 1, 489–530 DeDuve C & Baudhuin P (1966) Peroxisomes (microbodies and related particles) Physiol Rev 46, 323–357 Wanders RJ (2004) Metabolic and molecular basis of peroxisomal 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Traffic 4, 491–501 FEBS Journal 272 (2005) 2362–2372 ª 2005 FEBS ... for the import of PTS1 and PTS2 proteins still import peroxisomal membrane proteins Thus, the import of peroxisomal membrane and matrix proteins is independent [41,42,72] The peroxisomal membrane. .. bind cargo proteins in the cytosol and direct them to a docking and translocation complex at the peroxisomal membrane There, the cargo is released and translocated across the peroxisomal membrane. .. into peroxisomes In the absence or deficiency of peroxisomal membranes, these proteins might be imported into the reticular structures and contribute to the de novo synthesis of peroxisomes Whether