The discovery of the peroxisomal ATPase Pex1p triggered the beginning of the research on AAA (ATPase associated with various cellular activities) proteins and the genetic dissection of peroxisome biogenesis. Peroxisomes are virtually ubiquitous organelles, which are connected to diverse cellular functions. The highly diverse and adaptive character of peroxisomes is accomplished by modulation of their enzyme content, which is mediated by dynamically operating protein-import machineries. The import of matrix proteins into the peroxisomal lumen has been described as the ATP-consuming step, but the corresponding reaction, as well as the ATPase responsible, had been obscure for nearly 15 years. Recent work using yeast and human fibroblast cells has identified the peroxisomal AAA proteins Pex1p and Pex6p as mechano-enzymes and core components of a complex which dislocates the cycling import receptor Pex5p from the peroxisomal membrane back to the cytosol. This AAA-mediated process is regulated by the ubiquitination status of the receptor. Pex4p [Ubc10p (ubiquitin-conjugating enzyme 10)]-catalysed mono-ubiquitination of Pex5p primes the receptor for recycling, thereby enabling further rounds of matrix protein import, whereas Ubc4p-catalysed polyubiquitination targets Pex5p to proteasomal degradation.
- ATPase associated with various cellular activities (AAA)
- protein transport
Pex1p (formerly Pas1p), Sec18p [NSF (N-ethylmaleimide-sensitive factor)] and Cdc48p (cell division cycle 48 protein) [p97/VCP (valosin-containing protein)] represent the first proteins that were recognized as belonging to a novel family of ATPases , the AAA (ATPase associated with various cellular activities) family , which later was extended to the family of AAA+ proteins . Belonging to the class of P-loop (phosphate-binding) NTPases, the AAA+ proteins are especially distinguished by the presence of at least one evolutionarily conserved 200–250-amino-acid ATP-binding domain that contains Walker A and B motifs in addition to other structural features, such as the SRH (second region of homology), which distinguishes AAA proteins from other AAA+ proteins . Although the members of the AAA+ family display a high functional diversity, the common function of all seems to be the ability to catalyse reactions that are associated with significant conformational remodelling of substrate proteins or nucleic acids .
A lot of detailed information regarding the structure and molecular mechanism of AAA proteins has been accumulated, but our understanding of the molecular function of Pex1p and Pex6p, the second AAA peroxin in peroxisomal biogenesis , remained incomplete for many years.
Peroxisomes are single-membrane-bound organelles of virtually all eukaryotic cells, which display a unique variability in their enzyme content and metabolic functions that are adjusted according to the cellular needs. Their matrix harbours at least 50 different enzymes that are linked to diverse biochemical pathways. The β-oxidation of fatty acids and the detoxification of hydrogen peroxide are regarded as the central function of peroxisomes. They are the source of signalling molecules such as jasmonates in plants or lipid-derived ligands for PPARs (peroxisomeproliferator-activated receptors) in humans. Other functions of peroxisomes include the final steps of penicillin biosynthesis in some filamentous fungi, the main reactions of photorespiration in leaf peroxisomes and the synthesis of bile acid and ether lipids such as plasmalogens in mammals, which contribute more than 80% of the phospholipid content of the white matter in the brain .
Because of the central role of peroxisomes in lipid metabolism, they are essential for normal human development and physiology. This is emphasized by a group of genetic disorders collectively referred to as the peroxisome disorders, which, in most cases, lead to death in early infancy . Detailed analysis of the complementation groups finally revealed that the most common cause of peroxisomal biogenesis disorders are mutations in Pex1p . More than 80% of all patients with Zellweger syndrome, the most severe peroxisome biogenesis disorder, carry mutations in Pex1p or Pex6p .
Molecular architecture of the peroxisomal AAA complex
AAA proteins are characterized by a typical modular architecture as they contain an N-terminal non-ATPase domain which is followed by at least one conserved AAA domain. Each AAA cassette usually contains an ATP-binding site (Walker A) and an ATP-hydrolysis site (Walker B) along with other motifs, such as the SRH .
Pex1p and Pex6p are type II AAA proteins, which are characterized by two AAA domains (Figure 1). In both AAA peroxins, the second AAA domain is more conserved than the first one. Interaction and subsequent oligomerization of Pex1p and Pex6p is believed to be initiated in the cytosol and involves their first less conserved AAA domains (D1) [11,12]. Although neither binding nor hydrolysis of ATP at D1 seems to be essential for functionality in both yeast and humans, the interaction of human Pex1p and Pex6p is stimulated by binding of ATP to D1 of human Pex1p and Pex6p [12,13]. Furthermore, ATP binding, but not hydrolysis, at the second AAA cassette (D2) of Pex1p is required for the Pex1p–Pex6p interaction in both systems [11,12].
Pex1p and Pex6p are believed to form heterohexameric structures in the cytosol and at the peroxisomal membrane [12,14–16]. However, it is not clear whether formation of a heteromeric assembly of the AAA peroxins is a prerequisite for their function, as one population of Pex1p does not co-localize with Pex6p in mammalian cells [12,17]. Although the formation of hexameric structures is common to AAA proteins, the formation of heterohexamers has been found in few other cases, such as the m-AAA (matrix AAA) complex, consisting of Yta10p and Yta12p, which is active at the matrix site of the inner mitochondrial membrane,  or the six different Rpt ATPases from the 19S proteasome .
The recruitment of AAA complexes to peroxisomes is mediated by the tail-anchored peroxisomal membrane proteins Pex15p in Saccharomyces cerevisiae or its functional orthologue Pex26p in human cells via binding of the N-terminal domain of Pex6p, stimulated by ATP binding to the Walker A motif of Pex6p D1 [20,21]. In contrast, the Walker A and B motifs of Pex6p D2 are required for an efficient detachment from Pex15p/Pex26p [12,20,22]. Although Pex15p and Pex26p have been described as adaptor proteins for the N-terminal part of Pex6p, no adaptor has yet been identified for Pex1p.
The NTD (N-terminal domain) of murine Pex1p represents the only available crystal structure of the AAA peroxins . The NTD folds into two structurally independent globular subdomains (N- and C-lobe), which comprise an N-terminal double-Ψ fold and a C-terminal β-barrel, separated by a shallow groove. Similar grooves were found in the adaptor-binding sites within the NTDs of VCP, NSF and VAT (VCP-like ATPase from Thermoplasma), suggesting functional similarity .
To conclude, at least in S. cerevisiae, the Pex1p-bound nucleotides seem to influence the Pex1p–Pex6p interaction, while the different nucleotide states of Pex6p regulate the dynamic Pex6p–Pex15p/Pex26p association. The non-conserved domains are responsible for oligomerization, while the conserved domains exhibit the main ATPase activity.
Pex1p and Pex6p: peroxins associated with diverse cellular activities?
Besides their involvement in peroxisomal biogenesis, the AAA peroxins have been suggested to carry out other functions as well. Human Pex6p has been reported to interact specifically with the nucleocytoplasmatic transcriptional regulators Smad2, Smad3, Smad4 and Smad7 . These proteins are involved in the signalling pathway of the plasma membrane receptor TGFβ (transforming growth factor β), which regulates apoptosis. Furthermore, a suppressor screen for aging defects in mitochondria revealed that Pex6p, but not Pex1p, complements an ATP2-caused import defect into mitochondria, indicating a novel, yet not understood, function of this peroxin in mitochondrial inheritance and senescence .
In the context of peroxisomal biogenesis, the different functions discussed are mostly linked to the modulation of membrane dynamics. On the basis of the finding that Pex1p and Pex6p can associate with membranous subcellular structures distinct from mature peroxisomes in the yeasts Pichia pastoris and Yarrowia lipolytica, these peroxins were thought to play a role in lipid or membrane transport [14,26]. Utilizing in vitro fusion experiments, Pex1p and Pex6p were shown to be required for the fusion of five different premature peroxisomal vesicle species in Y. lipolytica , a process which might play a role during the maturation of endoplasmic reticulum-derived peroxisomal structures during de novo synthesis of peroxisomes . The still putative functional relevance of the observed phospholipid-binding activity of the murine Pex1p NTD, which has also been described for VCP and NSF, might be linked to this process . Furthermore, the presence of Pex6p and Pex15p is required for peroxisomal localization of the GTPase Rho1p, which is thought to organize actin filaments on peroxisomes during proliferation .
The existence of a link between the AAA peroxins and matrix protein import has been proposed previously , but has remained elusive for many years. Recently, their functional role in peroxisomal protein import was discovered. The AAA peroxins are required for the dislocation of the cycling peroxisomal import receptors Pex5p and Pex20p from the peroxisomal membrane back to the cytosol in order to complete their receptor cycle [31–34].
The AAA peroxins function as dislocases for the ubiquitinated PTS1 (peroxisomal targeting signal 1) receptor Pex5p
Import of folded proteins into peroxisomes occurs in a post-translational manner and depends on ATP. The soluble PTS1 receptor Pex5p is the major signal-recognition factor of proteins destined for the peroxisomal matrix. The receptor cycle of Pex5p involves cargo recognition in the cytosol, docking of the receptor–cargo complex to the peroxisomal membrane, translocation of the receptor–cargo complex to the luminal side of the membrane, followed by release of the cargo into the matrix and retrotranslocation of the receptor back to the cytosol (Figure 2) .
Permeabilized cell systems of human fibroblasts provided the first evidence that Pex5p accumulated reversibly at the peroxisomal membrane under ATP-modulated conditions . Detailed in vitro studies revealed that the binding and translocation of Pex5p itself is ATP-independent while the export of Pex5p back to the cytosol requires ATP . The identity of the corresponding ATPase remained a matter of debate until in vitro systems in S. cerevisiae  and human fibroblast cells  identified Pex1p and Pex6p as the motor proteins of Pex5p export. Their function in this process requires the presence of their membrane-anchor proteins, Pex15p or Pex26p. The in vitro reconstitution of the complete Pex5p cycle revealed that ATP binding and hydrolysis at both Pex1p D2 and Pex6p D2 is needed for receptor dislocation . Interestingly, the Walker B motif of Pex1p D2 seems to have no function in formation or targeting of the AAA complexes [11,12] and thus may be exclusively required for handling of the substrate. The binding and consumption of ATP is believed to induce conformational changes within the AAA peroxins that generate the driving force to pull the receptor out of the membrane by a mechanism possibly similar to the one of Cdc48p (p97/VCP) in ERAD (endoplasmic reticulum-associated degradation) .
The mechanism of substrate recognition by the AAA peroxins is not understood. Although Pex5p and the AAA proteins form a complex at the peroxisomal membrane [15,32,34], no direct interaction of the PTS1 receptor with either Pex1p or Pex6p has been reported. This interaction seems to be regulated or mediated by a third factor, which could represent an unknown adaptor protein of the AAA peroxins or post-translational modification of the substrate. It is well known that both processes play a central role in the function of Cdc48p (p97/VCP) [37,38], which is the closest evolutionary relative of Pex1p and Pex6p [39,40]. As a consequence, the question has to be addressed of how the AAA peroxins can distinguish Pex5p forms destined for dislocation from cargo-loaded Pex5p species destined for cargo translocation. A possible solution may arise from the crystal structure of Pex1p NTD, which displays similarities to the corresponding adaptor-binding domains of other AAA proteins . Data from p97 and Ufd1 have identified a double-Ψ β-barrel fold as a ubiquitin-binding domain with binding sites for both mono- and poly-ubiquitin .
Most interestingly, the PTS receptors Pex5p, Pex18p and Pex20p have been demonstrated to be ubiquitinated [31,42–44]. The PTS1 receptor Pex5p of S. cerevisiae is mono-ubiquitinated in wild-type cells , whereas it has been shown to be polyubiquitinated in mutants of the proteasome or cells affected in the AAA and Pex4p–Pex22p complexes of the peroxisomal protein-import machinery [42,43]. Polyubiquitination of Pex5p, requiring the ubiquitin-conjugating enzymes Ubc4p and the partly redundant Ubc5p and Ubc1p, takes place exclusively at the peroxisomal membrane and marks the receptor for proteasomal degradation as part of a quality-control system [42,43,45]. Alternatively, Pex5p is the specific molecular target for mono-ubiquitination by Pex4p (Ubc10p) [33,46], which is essential for peroxisomal biogenesis  and is anchored via Pex22p to the peroxisomal membrane .
The functional role of ubiquitination in the dislocation process has been elucidated by in vitro export assays, revealing that mono-ubiquitination of Pex5p constitutes the export signal under physiological conditions, whereas polyubiquitination seems to provide an export signal for the release of dysfunctional PTS1 receptors from the membrane and proteasomal degradation as part of the quality-control pathway .
The direct mechanistic influence of this modification on the export reaction remains to be investigated. The AAA peroxins may interact directly or indirectly via putative adaptors with the ubiquitin tag on Pex5p. Alternatively, the attachment of ubiquitin may induce local conformational changes within Pex5p to expose hidden binding sites. This mode of interaction is also discussed for Cdc48p (p97/VCP), which binds ubiquitin via adaptor complexes such as Ufd1/Npl4 and via its N-terminal domain. This domain is capable of recognizing ubiquitin chains and also non-modified segments of its substrates [49,50].
Notably, the AAA complex displays significantly increased association with the importomer in PEX4-deficient cells, indicating that the ATPase cycles of Pex1p and Pex6p are coupled to the mono-ubiquitination-dependent receptor cycle of Pex5p (Figure 1) .
Peroxisomes exhibit unique dynamics in their enzyme content and metabolic functions. The accompanied changes are accomplished by elaborate protein-transport machineries. The energy requirement for peroxisomal protein import is determined by the ATP-dependent dislocation of the import receptors, which probably represents the rate-limiting step. The energy is utilized by two enzyme activities: (i) mono-ubiquitination by Pex4p (recycling pathway) or polyubiquitination by Ubc4p (proteolytic pathway), as ubiquitin first has to be activated by E1; and (ii) ATP hydrolysis in the conserved AAA domains of Pex1p and Pex6p in order to pull the primed PTS receptor out of the membrane.
These results bring together the previously disparate roles of Pex4p and the AAA peroxins in one concerted reaction sequence. For future research, it will be a challenge to elucidate how AAA-mediated receptor dislocation is mechanistically linked to the peroxisomal import of folded proteins.
Note added in proof (received 13 December 2007)
After submission of the present paper, an article appeared concerning the ubiquitination of mammalian Pex5p . This study demonstrates that this modification is required for recycling and thus reveals that the mechanism of AAA peroxin function is highly censerved in evolution.
We apologize to those scientists whose work could not be cited due to space limitations. We are grateful to Sigrid Wüthrich for technical assistance and Wolfgang Girzalsky and Marion Witt-Reinhardt for the reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SFB642, Er178/2-4), the FP6 European Union Project ‘Peroxisome’ (LSHG-CT-2004-512018) and by the Fonds der Chemischen Industrie.
Seventh International Meeting on AAA Proteins: Independent Meeting held at the Royal Agricultural College, Cirencester, U.K., 9–13 September 2007. Organized and Edited by John Mayer (Nottingham, U.K.) and Paul Freemont (Imperial College London, U.K.).
Abbreviations: AAA, ATPase associated with various cellular activities; Cdc48p, cell division cycle 48 protein; NSF, N-ethylmaleimide-sensitive factor; NTD, N-terminal domain; PTS, peroxisomal targeting signal; SRH, second region of homology; Ubc, ubiquitin-conjugating enzyme; VCP, valosin-containing protein
- © The Authors Journal compilation © 2008 Biochemical Society