The AAA-type ATPases Pex1p and Pex6p and their role in peroxisomal matrix protein import in Saccharomyces cerevisiae

Biochimica et Biophysica Acta 1823 (2012) 150–158

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Review

The AAA-type ATPases Pex1p and Pex6p and their role in peroxisomal matrix protein import in Saccharomyces cerevisiae☆ Immanuel Grimm a, 1, Delia Saffian a, 1, Harald W. Platta b, Ralf Erdmann a,⁎ a b

Abteilung für Systembiochemie, Medizinische Fakultät der Ruhr-Universität Bochum, D-44780 Bochum, Germany Department of Biochemistry, Institute for Cancer Research, The Norwegian RadiumHospital, Montebello, N-0310 Oslo, Norway

a r t i c l e

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Article history: Received 17 June 2011 Received in revised form 2 September 2011 Accepted 3 September 2011 Available online 22 September 2011 Keywords: AAA-type ATPases PEX Peroxin Peroxisome biogenesis Protein translocation Ubiquitination

a b s t r a c t The recognition of the conserved ATP-binding domains of Pex1p, p97 and NSF led to the discovery of the family of AAA-type ATPases. The biogenesis of peroxisomes critically depends on the function of two AAAtype ATPases, namely Pex1p and Pex6p, which provide the energy for import of peroxisomal matrix proteins. Peroxisomal matrix proteins are synthesized on free ribosomes in the cytosol and guided to the peroxisomal membrane by specific soluble receptors. At the membrane, the cargo-loaded receptors bind to a docking complex and the receptor-docking complex assembly is thought to form a dynamic pore which enables the transition of the cargo into the organellar lumen. The import cycle is completed by ubiquitination- and ATP-dependent dislocation of the receptor from the membrane to the cytosol, which is performed by the AAA-peroxins. Receptor ubiquitination and dislocation are the only energy-dependent steps in peroxisomal protein import. The export-driven import model suggests that the AAA-peroxins might function as motor proteins in peroxisomal import by coupling ATP-dependent removal of the peroxisomal import receptor and cargo translocation into the organelle. This article is part of a Special Issue entitled: AAA ATPases: structure and function. © 2011 Elsevier B.V. All rights reserved.

1. Introduction In the early 90s, the recognition that Pex1p (formerly termed Pas1p), the first factor identified to play a role in the biogenesis of peroxisomes, shares a highly conserved ATP-binding domain with NSF (N-ethylmaleimide-sensitive factor) and VCP (valosin-containing protein) [1] led to classification of a new family of ATPases called AAA-type ATPases (ATPases associated with various cellular activities) [1–5]. As the name implies, AAA proteins operate in a broad range of cellular activities (reviewed by [6]). NSF (Sec18p in yeast) for instance, is essential for vesicular transport processes in eukaryotes [2,7] where it mediates the disassembly of SNARE (α-SNAP receptor) complexes and enables vesicular fusion [8]. Cdc48p (and its ortholog p97/VCP in mammals) is a multi-tasking protein that is required for

Abbreviations: AAA, ATPases associated with various cellular activities; Cdc48p, cell division cycle 48 protein; ERAD, endoplasmic reticulum associated degradation; NSF, N-ethylmaleimide-sensitive factor; NTD, N-terminal domain; Pex, peroxin; PTS, peroxisomal targeting signal; RING, really interesting new genes; Ubc, ubiquitin conjugating enzyme; VCP, valosin-containing protein ☆ This article is part of a Special Issue entitled: AAA ATPases: structure and function. ⁎ Corresponding author at: Institut für Physiologische Chemie, Ruhr-Universität Bochum, Universitätsstr. 150, D-44780 Bochum, Germany. Tel.: +49 234 322 4943; fax: +49 234 321 4266. E-mail address: [email protected] (R. Erdmann). 1 These authors contributed equally to this manuscript. 0167-4889/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2011.09.005

membrane fusion [9], processing of transcription factors [10,11], and also plays a crucial role in ERAD (Endoplasmic Reticulum Associated protein Degradation) by providing the force to remove polyubiquitinated, misfolded proteins from the endoplasmic reticulum [12–14]. In the cytosol, these ubiquitin marked proteins are directed to the 26S proteasome. The six Rpt AAA-proteins of the 19S proteasomal cap control an onward unfolding and entry of the proteins to the proteasomal degradation core [15,16]. Other AAA-complexes are located at the inner membrane of mitochondria (m-AAA-, i-AAA; [17,18]), acting as chaperones (Hsp100 family; [19]), disassemble ESCRT-III complexes (endosomal sorting complex required for transport) at endosomal membranes (Vps4; [20]) or participate in microtubule associated processes as motor proteins (cytoplasmic dynein [21,22]) or severing of these filaments (Fidgetin, Katanin, Spastin; [23–25]). They can be found in bacteria as well, like FtsH, which combines dislocase and protease activities and functions in the quality control of bacterial membrane proteins [26]. Although AAA-type ATPases operate in such a multitude of cellular mechanisms, their molecular organization and functional mechanisms have striking similarities. A common principle is the ability to transfer the energy of ATP-hydrolysis to their target proteins, leading to extensive conformational changes such as unfolding or complex disassembly [27]. Furthermore, a variety of adaptor proteins control both the specific localization of the AAA-proteins as well as the energy transfer to their target proteins [28]. In this review, we focus on the AAA-type ATPases Pex1p and Pex6p, which are structurally related to Cdc48p/p97 and NSF. Pex1p and Pex6p are essential for

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biogenesis of peroxisomes in all eukaryotes. Proteins that play a role in peroxisome biogenesis are collectively called peroxins and mutations in the peroxin Pex1p are the most common cause for fatal peroxisome biogenesis disorders in human (see accompanying article by Fujiki and coworkers). The homologous proteins Pex1p and Pex6p form a complex which shuttles between the cytosol and the peroxisomal membrane. At the peroxisomal membrane, AAA-peroxins are responsible for the dislocation of the PTS1 (peroxisomal targeting signal 1) receptor Pex5p back into the cytosol and thus complete the peroxisomal matrix protein import cycle [29,30]. A similar role for the AAA-peroxins has been suggested for the export of the PTS2 co-receptor Pex20p in Pichia pastoris [31]. In this review, the emphasis is on Pex1p and Pex6p from yeast, especially Saccharomyces cerevisiae. 2. Architecture of the peroxisomal AAA-type ATPases AAA-proteins belong to the class of P-loop NTPases defined by conserved motifs for NTP-binding (Walker A motif) and hydrolysis (Walker B motif) which are assisted by Mg 2+ as cofactor [32]. Classification of the AAA-family is based not only on evolutionary conserved AAA-domains comprising 200–250 amino acids, which contain the Walker A and B motifs as well as other conserved regions like the second region of homology (for details see accompanying reviews of Zhang and coworkers and Wendler and coworkers). The identification of additional ATPases possessing AAA-modules led to an extension of the protein family designated as AAA +-ATPases [4,33–35]. This expanded family includes further metabolically active and transcriptionally regulated proteins together with atypical AAAproteins such as cytoplasmic Dynein [36] (see accompanying review by Cho and Vale). Type-I AAA-proteins, like Vps4p (see accompanying review by Hill and Babst) proteasomal ATPases (see accompanying review by Bar-Nun and coworkers) as well as microtubule severing AAA-proteins (see accompanying review by Reid and coworkers), are characterized by a single AAA-module besides their N-terminal domain (NTD). The previously mentioned proteins NSF (see accompanying review by Whiteheart and coworkers), Cdc48p (see accompanying reviews by Ogura, Dargemont and Wolf and their coworkers) as well as Pex1p and Pex6p belong to the group of type-II AAA proteins. These proteins are characterized by the presence of two AAA-domains, termed D1 and D2, post positioned to an N-terminal domain (NTD) (Fig. 1). The Walker A- and B-motifs for ATP-binding and ATP-hydrolysis of the D1-domain have been named A1 and B1, the Walker motifs for the D2-domain accordingly A2 and B2. The AAA-domains in Cdc48p, NSF and the AAA-peroxins exhibit different degrees of conservation. While both AAA-domains of Cdc48p are well conserved, only the first AAA-domain (D1) of NSF and only the second AAA-domain (D2) of the AAA-peroxins are evolutionary conserved. A common feature of classical AAA-proteins is the formation of active oligomers with predominantly hexameric constitution [34]. Thereby the AAA-domains are arranged in a ring-shaped pattern, surrounding a central cavity. This hexameric architecture is also observed for p97, based on X-ray crystal analysis [37–39] and cryoelectron microscopy [40–44]. Structural data also provided some insight into the reaction cycle of this ATPase. Binding of ATP and its hydrolysis result in conformational changes of the D1 and D2 domains, leading to overall structural changes, whereby the shift of the central cavity from an open to a closed state is most conspicuous [41]. Our knowledge on the structural arrangement and the functional mechanism of the AAA peroxins Pex1p and Pex6p is still scarce. However, their close relationship with Cdc48p/p97 and NSF [45,46] suggests that they might share a similar mode of action. Of all known AAA-peroxins, only the N-terminal domain (NTD) of murine Pex1p has been crystallized and its structure has been solved. It consist of two globular subdomains (N- and C-lobe) folded to an N-terminal double psi and a C-terminal beta-barrel. These subdomains are connected by a short linker and form an overall structure with a shallow groove similar

Fig. 1. Organization of the AAA-peroxins at the peroxisomal membrane in S. cerevisiae. The peroxins Pex1p and Pex6p are type-II AAA-proteins, which both comprise an N-terminal domain (NTD), a non-conserved AAA-domain (D1) and a conserved AAA-domain (D2). Each of these AAA-domains contains a Walker A motif (A1; A2) for ATP binding and, with the exception of D1 of Pex6p, a Walker B motif (B1, B2) for hydrolysis of ATP. The formation of a heteromeric AAA-complex is achieved by interaction of the D1 domains of Pex1p and Pex6p (blue arrow) and is stimulated by ATP binding to the A2 motif of Pex1p (indicated in blue). Recruitment of the AAA-complex to peroxisomes occurs via binding of the NTD of Pex6p to the N-terminus of the tail-anchored membrane protein Pex15p (green arrow). This interaction is stimulated by ATP binding to the Pex6p A1 motif (indicated in green). Detachment of the AAA-complex from Pex15p (red arrow) depends on the function of the D2 domain of Pex6p (indicated in red).

to the N-terminal domains of p97/VCP or NSF [47]. Interestingly, the NTDs of p97/VCP and Pex1p show a common phospholipid-binding activity, which may support the attachment to their target membranes [48]. While no adaptor proteins interacting with Pex1p have yet been identified, the NTD of Pex6p has been demonstrated to interact with the tail-anchored membrane protein Pex15p, for which orthologues have been identified in mammals (Pex26p) and plants (APM9) [49– 51]. Since Pex1p and Pex6p interact with each other [52], they are believed to form a heteromeric complex which is recruited via the Pex6p-Pex15p (Pex26p/APM9) interaction to the peroxisomal membrane. 3. The AAA-peroxins Pex1p and Pex6p are essential for peroxisome biogenesis The AAA proteins Pex1p and Pex6p are peroxisomal biogenesis factors, so called peroxins [1,53]. To date thirty-four peroxins are known to be required for the formation of peroxisomes [54,55]. They are involved in the key stages of peroxisomal biogenesis like the formation of the peroxisomal membrane, peroxisome proliferation as well as compartmentalization of peroxisomal matrix proteins. The biogenesis and function of peroxisomes strongly depend on the import of their matrix content. As these organelles neither contain DNA nor an own translation system, all proteins have to be imported after their synthesis on free ribosomes in the cytosol. Peroxisomes are ubiquitously present in eukaryotic cells, whereby their morphology and enzyme content differs, depending on species, tissue and environmental conditions. More than 100 different matrix proteins are known to traverse the single peroxisomal membrane to

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reach their destination where they fulfill a multitude of anabolic and catalytic functions. Typical and eponymous for peroxisomes are hydrogen peroxide producing oxidases coupled to the detoxification of H2O2 by catalases [56]. In yeast and most other organisms, betaoxidation of fatty acids exclusively takes place in peroxisomes [57]. In humans, fatty acids can be degraded by both peroxisomes and mitochondria with the peroxisomal β-oxidation being specialized for fatty acids like branched or very long chain fatty acids (VLCFA) [58–61]. Other crucial metabolic functions of human peroxisomes are their contribution to the synthesis of plasmalogens [62], bile acids [63] and cholesterol [64]. Mutations that result in defects in the formation of peroxisomes are the molecular cause for the devastating group of human diseases, the peroxisome biogenesis disorders. Dysfunction of components of the peroxisomal protein import machinery including Pex1p and Pex6p leads to the Zellweger syndrome spectrum (ZSS) of peroxisomal biogenesis disorders (PBDs) [65–67]. The ZSS consists of the Zellweger syndrome (ZS) [68,69], the neonatal adrenoleukodystrophy (NALD) [70] and the infantile Refsum disease (IRD) [71], which represent essentially the same disease with a more or less severe phenotype [72]. This group of diseases is inherited in an autosomal recessive manner, which manifests in severe neurologic, hepatic and renal abnormalities as well as dysmorphic facial features. In its most severe form, the ZS, affected persons die within the first year of life, often prenatal or shortly after birth [73–75]. Affected patient cell lines are characterized by the absence of morphologically detectable peroxisomes, which either are completely absent or present as empty membrane structures (”ghosts”), whereby matrix proteins are mislocalized to the cytosol [76,77]. Although mutations in several peroxins can cause ZSS, about 80% of all patients exhibit mutations in either PEX1 or PEX6. Mutations in PEX1 alone have been detected in more than half of all ZSS patients. Depending on the mutation within the complementation group this defect results in a milder (IRD or NALD) or severe (ZS) phenotype. The temperature sensitive PEX1 mutation G843D is the most common cause among complementation group 1. This results in a reduced Pex6p interaction and a drastically reduced steady state level of the peroxisomal import receptor Pex5p, most likely upon instability of the mutated Pex1p [78–80]. Interestingly, mutations of Pex26p cause peroxisomal biogenesis disorders of complementation group 8. Here a genotype–phenotype correlation could be shown as Pex26p acts as membrane anchor for Pex1p and Pex6p in mammals [81,82]. Therefore, not just the interaction between the AAA-peroxins, which is thought to be the most common cause for the ZSS [78], but also the recruitment to the peroxisomal membrane is a prerequisite for their function in peroxisome biogenesis (for a detailed review of the human AAA-peroxins see accompanying review by Fujiki and coworkers). 4. Assembly of the Pex1p/Pex6p-complex and recruitment to the peroxisomal membrane The peroxisomal Pex1p/Pex6p complex together with the mitochondrial Yta10p/Yta12p (see accompanying review by Langer and coworkers) and the six Rpt proteins of the proteasome (see accompanying review by Bar-Nun and coworkers) are the examples of heteromeric AAA-complexes. The interaction of Pex1p and Pex6p was originally observed in Pichia pastoris [83] and confirmed in many other species including Homo sapiens [84], S. cerevisiae [52,85], and Hansenula polymorpha [86]. Analysis of truncated and mutated versions of yeast Pex1p and Pex6p revealed that the interaction takes place via their first AAA-domains (D1) and is strongly enhanced by ATP-binding to the second AAA-domain (D2) of Pex1p [52] (Fig. 1). Compared with yeast, ATP-binding seems to be more important for the assembly of the AAA-complex of Pex1p and Pex6p in mammalian cells, since the Walker motifs A1 and A2 of both proteins are required for the interaction. In addition, ATP-hydrolysis in B1 of mammalian Pex1p is a prerequisite for an efficient assembly of the AAA-peroxins [52,87].

The Pex1p/Pex6p-complex shows a dual localization in the cell as it is located in the cytosol as well as at the peroxisomal membrane. Association of this complex with the peroxisomal membrane is mediated by binding to Pex15p. The predominant part of the tail-anchored protein Pex15p or its orthologues in man (Pex26p, [51]) or plants (APM9, [50]) faces the cytosol and mediates the peroxisomal membrane association of the AAA-complex via a direct interaction with the N-terminal domain of Pex6p [49,50,52,88]. However, the AAAdomains of Pex6p seem to influence the Pex6p/Pex15p-interaction and thereby regulate the recruitment of the cytosolic AAA-complex to the peroxisomal membrane, although in opposite fashion. In particular, ATP-binding to D1 of Pex6p stimulates association of the AAA-complex with Pex15p at the peroxisomal membrane [49] while ATP-hydrolysis at D2 seems to trigger the release of the AAA-complex from Pex15p and thus from the membrane [49]. In this context, it is noteworthy that the D1 of Pex6p of all species does not contain a functional Walker B motif for ATP hydrolysis [52,89]. The data indicate that the assembly and disassembly of the AAAcomplex as well as its Pex15p-mediated association and release from the membrane are dynamic processes regulated by the nucleotide binding state of the proteins. Studies of the mammalian AAAperoxins additionally demonstrated that recruitment of the AAAcomplex to peroxisomes is regulated by conformational changes during the ATPase cycle [90] (see also accompanying review by Fujiki and coworkers). AAA-peroxins defective in ATP-hydrolysis of D1 are at least partially functional. In contrast, ATP-hydrolysis of the conserved AAAdomains (D2) of both AAA-peroxins is essential for their function [52]. A picture is emerging in which Pex1p and Pex6p assemble via the less conserved D1 domains. The resulting Pex1p/Pex6p-complex associates with the peroxisomal membrane by binding of the Ndomain of Pex6p to the peroxisomal Pex15p. A functional role for the N-domain of Pex1p has not yet been unveiled. The conserved D2-domains of Pex1p and Pex6p require hydrolysis of ATP for their function in peroxisome biogenesis, indicating that they may provide the driving force for conformational changes triggered by the AAAperoxins. 5. Function of Pex1p and Pex6p in peroxisomal matrix protein import Pex15p connects the complex of Pex1p and Pex6p to the protein import machinery which assembles into a supramolecular complex at the peroxisomal membrane [85]. Impressive characteristics of the peroxisomal protein import machinery are (i) that the peroxisomal import receptors for cargo proteins shuttle between the cytosol and the peroxisomal membrane [91,92], (ii) that the cargo proteins are transported in a folded, even oligomerized manner with the basic mechanism of this process still being enigmatic [93,94] and (iii) that the AAA-peroxin dependent release of the import receptors from the membrane seems to be responsible for the overall energy requirement of the import process, which gives rise to the export-driven import model [95]. This concept proposes that the ATP-dependent dislocation of the peroxisomal import receptor by the AAA-ATPases is coupled to protein translocation into the organelle. As first step of the peroxisomal protein import cascade, newly synthesized proteins, designated for the transport to the peroxisomal matrix, are recognized in the cytosol by PTS-receptors. In the following, the cargo-receptor complex docks at the peroxisome, where the cargo proteins are transferred across the membrane, and are finally released into the peroxisomal lumen (Fig. 2). Most peroxisomal matrix proteins carry a PTS1 at their C-terminus which is recognized by the PTS1-receptor Pex5p in the cytosol [96,97]. The receptor Pex7p facilitates the import of proteins carrying a PTS2, depending on co-factors, such as Pex18p and Pex21p in S. cerevisiae or Pex20p

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Fig. 2. Peroxisomal matrix protein import in S. cerevisiae. The peroxisomal matrix protein import is based on soluble receptors, which shuttle between the cytosol and the peroxisomal membrane, thereby guiding cargo proteins to the peroxisomal translocation machinery. (I) The receptor Pex5p recognizes matrix proteins via their PTS1 in the cytosol, and (II) ferries them to the docking complex (Pex13p, Pex14p, Pex17p) at the peroxisomal membrane. Assembly of the cargo-loaded Pex5p with the docking complex is supposed to result in the formation of a transient pore, whose exact molecular organization is still under discussion but at least contains Pex5p and Pex14p. (III) The cargo is translocated into the peroxisomal lumen in an unknown manner, possibly remaining associated with its receptor. Then the receptor-cargo complex dissociates and the cargo is released into the peroxisomal lumen, a process which possibly involves Pex8p. (IV) Monoubiquitination of the receptor is accomplished by the Pex22p-anchored ubiquitin-conjugating Pex4p in concert with the ubiquitin ligase Pex12p, which forms the RING complex together with the other ubiquitin ligases Pex2p and Pex10p. The ubiquitin modification serves as signal for the ATP-dependent dislocation of Pex5p from the peroxisomal membrane back to the cytosol. This process is performed by the AAA-peroxins Pex1p and Pex6p, which are anchored to the peroxisomal membrane via Pex15p. Removal of the ubiquitin is supposed to complete the receptor cycle and provides Pex5p for new rounds of import.

in other yeasts [31,98–101]. After its formation, the cargo-receptor complex docks at the peroxisomal membrane via interactions with the membrane proteins Pex13p and Pex14p [102–106]. It remained a matter of debate for a long time how the folded cargo proteins traverse the membrane. One suggestion was that the import receptor might be part of a dynamic import pore [107]. Indeed, the PTS1receptor Pex5p together with Pex14p turned out to represent the minimal unit for the import of the intraperoxisomal protein Pex8p [108] and they form a pore with features expected for a proteinconducting channel [109]. Moreover, the dynamic behavior and physical properties of this channel with a diameter of up to 9 nm appear to fulfill the criteria for the passage of folded proteins. After formation of the import pore and cargo translocation, the receptor returns back to the cytosol and in this way is recycled for another round of import. Early experiments with permeabilized fibroblasts displayed an ATP-dependent shuttling of Pex5p between the peroxisomal membrane and the cytosol [91]. Interestingly, docking of the cargo-receptor complex to the peroxisomal membrane appeared to be ATP-independent, while the release of Pex5p from the organelle membrane required ATP hydrolysis [110]. Further studies with S. cerevisiae [30] and human fibroblast cells [29] identified that the AAA-type ATPases Pex1p and Pex6p are responsible for the release of Pex5p from the membrane. To fulfill this dislocase function, the AAA-peroxins rely on the linkage to the import machinery via binding to their membrane anchor Pex15p (Pex26p). Moreover, functional D2 domains of both Pex1p and Pex6p are required to shuttle the receptor back to the cytosol. As the Walker B motif of the D2 domain of Pex1p is necessary for receptor release but not for assembly of the AAA-complex and membrane recruitment [52,87], ATPhydrolysis in this domain may exclusively be required for receptor release. It is anticipated that an ATP-driven conformational change in the D2-domains of the AAA-peroxins is transferred to the import

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receptor to pull the protein out of its membranous environment. A fraction of the membrane-bound receptor is carbonate-resistant and therefore behaves like an integral membrane protein. It should be noted that also the integral receptor is released from the membrane by the AAA-peroxins [30]. We believe that the AAA-peroxins generate a pulling force which liberates the receptors from their membranebound state. Another question concerns substrate recognition, in particular how the AAA-peroxins interact with the import receptor Pex5p. Among other proteins of the import machinery, Pex5p was identified as component of solubilized AAA-complexes from yeast [30,85]. As direct binding between the AAA-peroxins and the PTS-receptor has not yet been detected, an interaction via adaptor proteins and/or posttranslational modifications of the involved proteins is considered. As an adaptor has not been identified, the attention focused on the discovery of ubiquitinated forms of Pex5p [111–113]. Ubiquitination of Pex5p takes place exclusively at the peroxisomal membrane and monoubiquitinated as well as polyubiquitinated forms have been detected [112–114]. Also the co-receptors of the PTS2-receptor Pex7p, Pex18p in S. cerevisiae and Pex20p in P. pastoris, were found to be modified by ubiquitination [31,115]. The transfer of ubiquitin to its target protein is catalyzed via an enzyme cascade. First, an ubiquitin-activating enzyme (E1) catalyzes the ATP-dependent transfer of ubiquitin to a conjugating enzyme (Ubc or E2). In the next step, an ubiquitin-protein ligase (E3) facilitates the covalent binding of the ubiquitin to the substrate [116]. Monoubiquitination of the PTS1-receptor Pex5p requires the peroxisomal E2 enzyme Pex4p (Ubc5Ha/b/c in mammals) [117–119] together with the RING (Really Interesting New Gene) ligase Pex12p [120] (Fig. 3). Polyubiquitination of Pex5p depends on the E2 enzyme Ubc4p (or the partially redundant Ubc5p and Ubc1p) [112–114]. In addition, the RING ligases Pex2p and Pex10p have been implicated to play a role in this process [120,121] (Fig. 3). Monoubiquitinated Pex5p is detected at isolated membranes of wild-type S. cerevisiae upon treatment with N-ethylmaleimide (NEM), which is supposed to block deubiquitination [112], whereas polyubiquitinated forms of Pex5p can be detected in cells with defects in late steps of the import cascade like mutations in the AAA-peroxins, Pex4p or the proteasome [113,114]. The latter indicates that the polyubiquitinated forms of Pex5p are designated for proteasomal degradation. In vitro export studies revealed that ubiquitination of the import receptor Pex5p is a prerequisite for its AAA-peroxin dependent release from the membrane [118], indicating that the attachment of ubiquitin indeed represents the signal for the AAA-dependent initiation of the export step [118]. Thus, ubiquitination could possibly distinguish the export competent Pex5p from the Pex5p species associated with an active import pore [107]. In comparison, monoubiquitination of Pex5p appears to be the common export signal dedicated for the recycling pathway, whereas polyubiquitination seems to be part of a quality control system to remove dysfunctional Pex5p from the membrane for proteasomal degradation. In vivo data from P. pastoris support the idea that ubiquitination also plays a role in cycling of receptors in the PTS2-pathway [31,122]. Pex20p is a coreceptor of the PTS2-pathway with sequence similarities to Pex5p. Block of Pex20p ubiquitination by mutagenesis of all ubiquitination sites results in its accumulation at the peroxisomal membrane. The data indicate that also Pex20p might undergo a similar cycling as the PTS1-receptor Pex5p, including its AAAperoxin dependent release from the membrane. After release of the monoubiquitinated receptor from the membrane, Pex5p is exclusively found as non-modified protein in the cytosol. This observation suggests that the receptor is deubiquitinated, which may take place either during the export process or shortly afterwards [123]. Interestingly, the monoubiquitin moiety is linked to a cysteine of Pex5p via an energetic weak thioester bond. Recent data indicate that deubiquitination of mammalian Pex5p

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Fig. 3. Ubiquitination cascades at the peroxisomal membrane in S. cerevisiae. The PTS1-receptor Pex5p is either mono- or polyubiquitinated at the peroxisomal membrane by two different ubiquitination cascades. Both cascades are ATP-dependently initiated by the ubiquitin-activating enzyme Uba1p. For the monoubiquitination of Pex5p (green arrows) the activated ubiquitin is transferred to the ubiquitin conjugating enzyme Pex4p and finally delivered to Pex5p facilitated by the RING-ligase Pex12p. After Pex1p/Pex6p-dependent export and removal of the monoubiquitin the receptor is provided for further rounds of import. Alternatively, activated ubiquitin is transferred from Uba1p to the ubiquitin conjugating enzyme Ubc4p, which acts in concert with the RING-ligase Pex2p on Pex5p polyubiquitination (red arrows). Since polyubiquitinated Pex5p is degraded by the 26S proteasome after its AAA-dependent translocation, this second ubiquitination cascade is considered to be part of a quality control system.

may be accomplished enzymatically or non-enzymatically by a nucleophilic attack of glutathione [123]. While in mammals deubiquitinating enzymes acting on Pex5p have not yet been identified, the ubiquitin-hydrolase Ubp15p has been linked to this process in yeast via its interaction to Pex6p [124]. Ubp15p is associated with the AAA-complex via a direct binding to Pex6p. Deficiency in Ubp15p results in accumulation of ubiquitinated receptor at the peroxisomal membrane and a partial import defect for peroxisomal matrix proteins, indicating that receptor deubiquitination might be an important process in receptor export and recycling [124]. It is interesting to note that Pex4p-mediated monoubiquitination of Pex5p might not only control the export of the modified receptor but may also contribute to the release of the AAA-peroxins from the membrane [119]. In Pex4p-deficient yeast Pex1p and Pex6p accumulate at the importomer [85]. This raises the possibility that the Ubdependent PTS1-receptor cycle and the recruitment and release of the AAA-peroxins are interconnected. 6. Pex1p/Pex6p and Cdc48p — common features, different tasks The Pex1p-Pex6p-complex and Cdc48p/p97 share striking similarities. Both are type-II AAA ATPases and have similar functions as they are crucial for the ATP-dependent translocation of proteins across a membrane. Cdc48p/p97 takes part in several cellular processes like membrane fusion, cell cycle progression and transcription factor processing and also acts as a major player in ERAD (see accompanying review by Wolf and coworkers). ERAD is responsible for the removal of misfolded ER-proteins and their subsequent degradation by the 26S proteasome in the cytosol [125,126]. Chaperones and enzymes like protein disulfide isomerases in the ER lumen assist proper folding and modifications of newly imported ER-proteins. Proteins in the ER-lumen or the ER-membrane, which fail to achieve their natural conformation, are recognized and eliminated by ERAD. This requires retrotranslocation of luminal proteins or extraction in the case of membrane proteins. At the cytosolic face of the ER-membrane, these proteins are polyubiquitinated and degraded by the 26S proteasome [125]. Thus, ERAD is regarded as a protein quality control system. At first glance, the matrix protein import into peroxisomes and the extraction of misfolded substrates out of the ER membrane seem to be two different mechanisms. In case of the peroxisomal matrix protein import, proteins are transported from cytosol to peroxisomal lumen whereas proteins are released from the ER lumen

or membrane into the cytosol by ERAD. But a closer look at these machineries, both responsible for translocation processes, reveal a very similar protein composition and similar mode of action. The ERAD machinery as well as the peroxisomal import machinery consist of at least one channel for protein translocation (most likely Pex5p/ Pex14p for peroxisomes and Sec61p at least for a subset of ERAD substrates), membrane-anchored E2 enzymes (Pex4p/Pex22p at peroxisomes and Ubc7p/Cue1p in ERAD), several ubiquitin ligases (Pex2p/ Pex10p/Pex12p at peroxisomes and Hrd1p/Doa10p in ERAD) and an AAA-complex for the ATP-dependent extraction of the ubiquitinated substrate (Pex1p/Pex6p at peroxisomes and Cdc48p in ERAD) [95,127,128]. Ubiquitination as central signal and the action of AAA-ATPases as motor protein for the export process connects the mechanisms underlying both machineries and explains their similar composition. However, it has to be noted that ERAD substrates finally are always polyubiquitinated. In ERAD, substrates for ubiquitination are misfolded proteins destined for removal from the membrane and subsequent degradation. In peroxisomal protein import, it is the import receptor that is mono- or polyubiquitinated and exported back into the cytosol. Thus, in both cases the target of ubiquitination is transported to the cytosol, the ubiquitinated Pex5p receptor can be compared with an ubiquitinated ERAD substrate. Ubiquitination serves as export signal and is recognized by cytosolic AAA-complexes, which are recruited to their target membrane by a transmembrane protein serving as membrane anchor. In ERAD, Ubx2 and Dfm1 have been implicated to guide the AAA-complex to the ubiquitin ligases, where the ubiquitinated substrate is poised for translocation [129–133]. For ERAD, several models exist concerning the role of Cdc48p/p97 for the extraction of ER proteins [125]. Evidence has been provided that the proteasome itself can directly extract proteins from the ER membrane [134–136]. However, most proteins seem to require the activity of Cdc48p/p97 prior to the proteasome. Interestingly, Cdc48p has also been reported to function as a segregase in the release of the ubiquitinated transcription factor Spt23p from the ER membrane. Spt23p is then not degraded by the proteasome but transported into the nucleus [10,11]. For Cdc48p/p97, several models for recognition of the ubiquitinated target have been postulated: A direct binding to the substrate without involving ubiquitin [137], a direct interaction with ubiquitin [138], as well as a simultaneous binding to ubiquitin and a non-ubiquitinated part of the substrate [139]. Furthermore, an indirect interaction bridged by ubiquitin-binding adaptors has been described [140].

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Polyubiquitination and subsequent proteasomal degradation as part of a quality control system are common features of ERAD and peroxisomal matrix protein import. However, degradation of the import receptor Pex5p seems to be of importance if the protein import machinery is blocked but usually is not the main purpose of the AAA-peroxins. In fact, the Pex5p designated for degradation is marked by a polyubiquitin-chain at lysine-residues. For receptor recycling, Pex5p is modified by monoubiquitin of a conserved cysteine. Interestingly, both mono- and polyubiquitinated forms require the action of the AAA-peroxins for membrane release [118]. It is still unknown how the AAA-complex recognizes the marked receptor for export. The X-ray structure of the N-terminal domain of Pex1p showed a double-psi beta-barrel fold, which also is present in Cdc48p/p97 [47] and functions as specific binding domain for mono- and polyubiquitin in p97 [141]. But whether this domain performs such function in Pex1p still has to be investigated. Although our knowledge on complex composition and function is steadily increasing, the exact mechanism of the ATP-dependent extraction of protein substrates by Cdc48p/p97 as well as by the AAA peroxins, Pex1p and Pex6p still remains elusive and emphasis of future work will be on the elucidation of this process.

7. Other functions assigned to Pex1p and Pex6p Pex1p and Pex6p are evolutionary related to Cdc48p/p97 [45,127]. It is known that Cdc48p is not only involved in ERAD but also plays a crucial role in other cellular processes such as e.g. the homotypic fusion of ER membranes, assembly of Golgi cisternae, the processing of nuclear transcription factors or the expansion of the nuclear envelope [142]. Equally, Pex1p and Pex6p have been implicated in several different functions. Based on the finding that both Pex1p and Pex6p can associate with membranous subcellular structures distinct from mature peroxisomes in Yarrowia lipolytica and Pichia pastoris, these peroxins were thought to play a role in vesicular membrane transport [83,143]. By in vitro vesicle fusion experiments, Pex1p and Pex6p were shown to be required for the fusion of distinct premature peroxisomal vesicle species in Y. lipolytica [143]. The hypothesis has been put forward that this process might play a role during the maturation of endoplasmic reticulum-derived peroxisomal structures during de novo synthesis of peroxisomes [144]. Interestingly, Pex6p might also have additional functions that appear not to be related to peroxisomes. Human PEX6 has been reported to interact specifically with transcriptional regulators Smad2, Smad3, Smad4 and Smad7, which shuttle between endosomes, cytosol and nucleus [145]. These proteins are involved in the signaling pathway of the plasma membrane receptor TGFβ (transforming growth factor β), which regulates apoptosis. In addition, yeast Pex6p acts as a suppressor for aging defects in mitochondria [146]. Overexpression of Pex6p, but not of Pex1p, restores the import defect of mutant ATP2, the gene encoding the β-subunit of mitochondrial F1,F0-ATPase, into mitochondria. This finding might indicate a novel, yet not understood, function of this peroxin in mitochondrial inheritance and senescence [146]. Furthermore, a screen for aceticacid induced cell death revealed that the PEX6-deletion strain has the most pronounced survival defects of all strains affected in peroxisome function [147]. The study describes a function for Pex6p in the prevention of necrotic cell death in yeast. In summary, there is evidence that the AAA-peroxins might play distinct roles in vesicular membrane traffic and in the suppression of different cell death mechanisms. It will be of importance to analyze the functional role of the AAA-peroxins in these processes in greater detail and in different species. A common theme for the mode of action of the AAA-peroxins might be that they act as mechanoenzymes that disassemble protein complexes linked to different cellular functions.

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8. Conclusions and perspectives Ever since the basic set of components of the protein complexes dedicated to peroxisome function had been identified, the role of the AAA-type ATPases Pex1p and Pex6p has been the center of discussion regarding the molecular mechanism of this biogenetic process. While realizing that both AAA-peroxins might be involved in different tasks, several attempts to define their main contribution to peroxisome biogenesis have been made. First, mechanistic data on the function of Pex1p and Pex6p came from work in Y. lipolytica and linked both AAA-peroxins to fusion events of premature peroxisomal vesicle species [148]. A role for the AAA-type ATPases in matrix protein import was put forward by the hypothesis that they might disassemble peroxisomal cargo complexes on the cytosolic side of the membrane prior to the final import reaction (preimplex hypothesis) [149]. Another notion on the functional role of the AAA-peroxins in peroxisome biogenesis is related to the concept of a transient pore for peroxisomal protein import [107]. Here, the AAA-peroxins play an important role in receptor recycling. In particular, they function as dislocases for the ubiquitinated import receptors. Several groups working with different species have lent credibility to the concept that Pex1p and Pex6p consume ATP in order to extract Pex5p from the peroxisomal membrane. After release, monoubiquitinated Pex5p enters the recycling pathway for further rounds of import, while polyubiquitinated Pex5p is destined for proteasomal degradation. The AAA-dependent release of the import receptor seems to be responsible for the overall energy requirement of peroxisomal protein import. This is addressed by the export-driven import model, which describes a direct coupling of receptor export back to the cytosol and cargo release into the peroxisomal lumen [95,128]. Energetically driven by the peroxisomal AAA-proteins, a conformational change might be transferred to the receptor, which contributes to cargo translocation [95]. Alternatively, the cargo may be released from the receptor via competing binding affinities, most likely via the intraperoxisomal Pex8p. Thus, the AAA-dependent export of Pex5p might be the ratelimiting step in the protein import process. Probably cargo-free receptors have to be removed from the membrane in order to enable cargo-charged receptors to access the peroxisomal matrix [95,128]. The aim of future research will be to test these concepts in greater detail. In addition, it will be of importance to identify new adaptor proteins of Pex1p and Pex6p, which may possibly be specific to their functional implication in vesicle fusion, apoptosis and dislocation of the ubiquitinated PTS-receptors. Acknowledgements We apologize to all the scientists whose work could not be cited due to space limitations. We are grateful to Sigrid Wüthrich for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB642). DS was supported by the Research School of the Ruhr-University Bochum. HWP was supported by an EMBO Longterm Fellowship. References [1] R. Erdmann, F.F. Wiebel, A. Flessau, J. Rytka, A. Beyer, K.U. Fröhlich, W.-H. Kunau, PAS1, a yeast gene required for peroxisome biogenesis, encodes a member of a novel family of putative ATPases, Cell 64 (1991) 499–510. [2] M.R. Block, B.S. Glick, C.A. Wilcox, F.T. Wieland, J.E. Rothman, Purification of an N-ethylmaleimide-sensitive protein catalyzing vesicular transport, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 7852–7856. [3] K.U. Fröhlich, H.W. Fries, M. Rudiger, R. Erdmann, D. Botstein, D. Mecke, Yeast cell cycle protein CDC48p shows full-length homology to the mammalian protein VCP and is a member of a protein family involved in secretion, peroxisome formation, and gene expression, J. Cell Biol. 114 (1991) 443–453. [4] W.-H. Kunau, A. Beyer, T. Franken, K. Götte, M. Marzioch, J. Saidowsky, A. SkaletzRorowski, F.F. Wiebel, Two complementary approaches to study peroxisome biogenesis in Saccharomyces cerevisiae: forward and reversed genetics, Biochimie 75 (1993) 209–224.

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