Biochemical Society Transactions

Seventh International Meeting on AAA Proteins

Dynamic and functional assembly of the AAA peroxins, Pex1p and Pex6p, and their membrane receptor Pex26p involved in shuttling of the PTS1 receptor Pex5p in peroxisome biogenesis

Yukio Fujiki, Non Miyata, Naomi Matsumoto, Shigehiko Tamura


The peroxisome is a single-membrane-bound organelle found in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient PBDs (peroxisome biogenesis disorders), such as Zellweger syndrome. Two AAA (ATPase associated with various cellular activities) peroxins, Pex1p and Pex6p, are encoded by PEX1 and PEX6, the causal genes for CG (complementation group) 1 and CG4 PBDs respectively. PEX26, which is responsible for CG8 PBDs, codes for Pex26p, the recruiter of Pex1p–Pex6p complexes to peroxisomes. We recently assigned the binding regions between human Pex1p and Pex6p and elucidated the pivotal roles that the AAA cassettes, D1 and D2 domains, play in Pex1p–Pex6p interaction and in peroxisome biogenesis. ATP binding to both AAA cassettes of Pex1p and Pex6p was a prerequisite for the Pex1p–Pex6p interaction and peroxisomal localization, but ATP hydrolysis by the D2 domains was not required. Pex1p exists in two distinct oligomeric forms, a homo-oligomer in the cytosol and a hetero-oligomer on peroxisome membranes, with these possibly having distinct functions in peroxisome biogenesis. AAA peroxins are involved in the export from peroxisomes of Pex5p, the PTS1 (peroxisome-targeting signal type 1) receptor.

  • ATPase associated with various cellular activities (AAA)
  • peroxin
  • peroxisome biogenesis
  • peroxisome biogenesis disorder (PBD)
  • peroxisome-targeting signal type 1 receptor shuttling


Peroxisomes are single-membrane-bound organelles present in nearly all eukaryotic cells. The functional importance of peroxisomes in humans is highlighted by PBDs (peroxisome biogenesis disorders), including Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease and rhizomelic chondrodysplasia punctata, of which the primary cause is the impaired biogenesis of peroxisomes [1,2]. Genetic heterogeneity, consisting of 13 CGs (complementation groups), has been identified in PBDs [24] (Table 1). Human PEX1 and PEX6, which are responsible for PBDs of CG1 and CG4, were cloned by a functional complementation assay using CHO (Chinese-hamster ovary) cell mutants [57] and by an expressed sequence tag homology search using the yeast PEX1 and PEX6 genes [810]. Searches for pathogenic PBD genes were accomplished by functional cloning of PEX26, which is responsible for CG8 PBDs [3,11]. More than 30 PEX gene products, termed peroxins, are required for peroxisome assembly [2,12,13]. The biological functions of only a small number of peroxins have been delineated so far, including Pex5p and Pex7p, which are soluble receptors for proteins containing PTS1 (peroxisome-targeting signal type 1) and PTS2 respectively [14,15]. The roles and molecular mechanisms of most of the peroxins remain less well understood.

View this table:
Table 1 Complementation groups and complementing PEX genes of peroxisome deficiencies

*, Temperature-sensitive phenotype; IRD, infantile Refsum's disease; NALD, neonatal adrenoleukodystrophy; PMP, peroxisome membrane protein; PTSR-DP, PTS receptor-docking protein; RCDP, rhizomelic chondrodysplasia punctata; SH3, Src homology 3; TPR, tetratricopeptide repeat; ZS, Zellweger syndrome.

Peroxisomal matrix proteins are synthesized on free polyribosomes and are post-translationally imported into peroxisomes [16], requiring the concerted action of protein import machinery [17,18]. Pex1p and Pex6p are members of the large AAA (ATPase associated with various cellular activities) protein family involved in a wide range of different cellular processes, including vesicular transport, DNA repair, proteolysis and mitochondrial functions [1921]. One possible common functional feature of the AAA proteins is protein folding or unfolding in an ATP-dependent manner. AAA proteins share one or two AAA cassettes (known as D1 or D2 domains) that are characterized by a conserved sequence of 200–250 amino acids, which includes the Walker A and B motifs for ATP binding and ATP hydrolysis respectively [22,23]. Pex26p recruits Pex1p–Pex6p complexes to peroxisomes [3,24].

As a step to understanding the function of Pex1p and Pex6p in peroxisome biogenesis, we recently investigated the regions involved in the mutual binding and intracellular localization of Pex1p and Pex6p. Such interaction is likely to represent a relationship between peroxisomal localization and the functions of AAA peroxins. We also established an in vitro Pex5p-translocation system using peroxisomes isolated from CHO-K1 cells and from rat liver cells [25]. Pex5p was imported to and exported from peroxisomes in an ATP-independent and -dependent manner respectively. Moreover, Pex1p, Pex6p and Pex26p were likely to be dispensable for the import of Pex5p, but critical for Pex5p export. In the present paper, we discuss the shuttling of Pex5p between peroxisomes and the cytosol.

Dynamic and functional assembly of the AAA peroxins, Pex1p and Pex6p, and their membrane receptor Pex26p

We recently identified the regions involved in the interaction of human Pex1p and Pex6p [26]. We showed that two AAA cassette structures (D1 and D2 domains) are involved, and ATP binding to the two AAA cassettes of Pex1p and Pex6p is required for their association (Figure 1). In Saccharomyces cerevisiae, the interaction of Pex1p and Pex6p involves the respective first AAA cassette, requiring ATP binding, but not ATP hydrolysis in the second AAA cassette (D2) of Pex1p [27]. In contrast with this yeast system, the interaction of human Pex1p and Pex6p requires ATP binding to both D1 and D2 domains, suggesting that the interaction is enhanced by conformational changes in both the D1 and D2 domains on ATP binding [26].

Figure 1 A schematic model for Pex1p–Pex6p interaction and ternary complex formation with Pex26p

Cytosolic Pex1p is present mostly in a homo-trimer and partially in a hexamer. Pex1p oligomers are assembled into oligomer complexes with Pex6p. Walker motifs (A1, B1 and A2) of the AAA domain are essential for the interaction between Pex1p and Pex6p and their localization to peroxisomes (+), whereas the Walker B2 motif is dispensable (−). In contrast, ATP hydrolysis by Pex1p Walker B2 motif is required for catalase import, and the Pex6p Walker B2 motif initiates the release of Pex6p from Pex26p.

We reported previously that the dysfunction of PEX26 is responsible for PBDs of CG8 [3,11], and that Pex26p recruits Pex1p–Pex6p complexes to peroxisomes via Pex6p [3]. We also demonstrated the importance of the A1, B1 and A2 Walker motifs in Pex1p, and A1 and A2 Walker motifs in Pex6p, for their peroxisomal localization, which is in good agreement with the discovery of the Pex1p–Pex6p hetero-oligomer in binding assays [26]. Through morphological analysis, we found that neither Pex1p nor Pex6p were localized to peroxisomes in the absence of its mutual partner, thereby suggesting that the localization of Pex1p and Pex6p on peroxisomes requires the formation of ternary complexes with Pex26p in vivo. On the other hand, we reported previously that recombinant Pex6p bound to Pex26p fused to glutathione transferase in a pull-down assay [3]. Furthermore, we demonstrated in mammalian two-hybrid assays that Pex6p acts as a bridge between Pex26p and Pex1p, and that Pex26p bound to Pex6p in the absence of Pex1p [26]. Pex26p tagged with a nuclear localization signal bound to Pex6p in the nucleus [28]. Together, these results raise several possibilities with respect to the relationship between the ternary complexes and peroxisomal localization. Pex1p may stabilize Pex6p by forming the ternary complex on peroxisomes. It is also possible that Pex1p and Pex6p may interact with other peroxins that regulate the subcellular localization and function of AAA peroxins.

Pex1p, Pex6p and Pex26p are predominantly localized to peroxisomes on co-expression in pex26 ZP167 cells [26]. Pex1p is also detected in the cytosolic fraction of HEK-293 cells (human embryonic kidney cells), suggesting that Pex1p is present in the cytosol without interacting with Pex6p and Pex26p. Therefore the Pex1p–Pex6p interaction is likely to be indispensable for the peroxisomal localization of these proteins. Despite the failure to localize to peroxisomes as a result of the impaired interaction with Pex6p, several Pex1p mutants harbouring mutations in the Walker motifs A1 and B1 partially restored the peroxisomal import of PTS1 proteins and catalase, with nearly 50% efficiency when compared with wild-type Pex1p [26]. Recently, we reported several PBD patient-derived Pex26p mutants which showed insufficient binding to Pex1p–Pex6p complexes [24], hence inferring that Pex1p was likely to be present in the cytosol. Such Pex26p mutants were apparently competent in PTS1 import in the patients' fibroblasts. Taken together, a proportion of Pex1p in the cytoplasm is responsible for the transport of PTS1 proteins, but not catalase. With regard to the second AAA cassette (D2) of Pex1p, ATP binding was indispensable for the transport of both PTS1 proteins and catalase, whereas ATP hydrolysis was essential for the import of catalase. We reported that the import of PTS1 protein appeared to be normal in fibroblasts from PEX1-defective CG1 patients with infantile Refsum's disease, whereas the import of catalase and PTS2 proteins was impaired at 37°C [29,30]. At the permissive temperature (30°C), catalase and PTS2 import was normal. Therefore it is likely that the ATP-hydrolysing activity of Pex1p variants differentiates between the two distinct types of protein import.

Pex1p shows dynamic conformational changes in the presence or absence of Pex6p and Pex26p [26]. Pex1p forms two distinct oligomeric structures on the peroxisomal membrane or in the cytoplasm. As depicted by Blue Native PAGE analysis of cytosolic Pex1p from HEK-293 cells, Pex1p is mostly present as part of a homo-trimer, and less as a homo-hexamer. These results are similar to the findings in the structural study on NSF (N-ethylmaleimide-sensitive fusion protein) [31,32], another member of the AAA protein family. NSF forms cylindrical homo-oligomeric complexes, the ATP hydrolysis activity of which is essential for membrane fusion. Pex1p and Pex6p were earlier shown to be required for the fusion of peroxisomal membranes in the yeast Yarrowia lipolytica [33].

The N-terminal part of Pex6p provides the binding site for Pex26p, and ATP binding to D1 and D2 is required for stabilizing the interaction of Pex6p and Pex26p [26]. On the other hand, ATP hydrolysis by D2 is indispensable for Pex6p to dissociate from Pex26p. Recent reports [25,34] suggested that Pex1p and Pex6p are involved in the regulation of the translocation cycle of Pex5p, including the export step from peroxisomes (see below). Nevertheless, there is a functional analogy between Pex1p/Pex6p and NSF, at least in mammalian cells. The AAA family of peroxins is likely to play multiple roles in peroxisome biogenesis.

Shuttling mechanism of PTS1 receptor Pex5p: ATP-independent import and ATP-dependent export

According to the PTS1-receptor recycling model [15,17,18], cargo-loaded Pex5p targets peroxisomes, translocates across the peroxisomal membrane, unloads cargo and finally exits back to the cytosol. This protein import process has been thought to require several peroxisomal peroxins, such as Pex14p, Pex13p, Pex2p, Pex10p and Pex12p. The components involved at each step of the whole processes remain elusive.

We recently established a cell-free system for examination of Pex5p translocation (Figure 2) [25]. By making use of wild-type and several pex mutant CHO cells, we investigated the dynamism of Pex5p, including its import to and export from peroxisomes, as well as Pex5p translocation steps through potential import machinery complexes on peroxisomal membranes. Pex5p was specifically imported into peroxisomes from both CHO-K1 and rat liver cells. Pex5p did not bind to the peroxisome remnants of Pex14p-deficient ZP161 cells, demonstrating that Pex14p is the initial Pex5p-docking peroxin on peroxisomes, consistent with our earlier morphological and biochemical findings [35]. We also showed that ATP was not required for the import of Pex5p. Nevertheless, Pex5p import was temperature-sensitive, implying that Pex5 translocation across the peroxisomal membrane is driven by thermodynamic energy. This ATP-independent translocation is distinct from import in mitochondria and endoplasmic reticulum, where proteins are imported in an ATP-dependent manner [36,37]. We also developed an in vitro Pex5p export system [25] (see Figure 2). Using this system, we demonstrated that the Pex5p imported into peroxisomes was exported back to the cytosol in an ATP-dependent manner, in sharp contrast with the ATP-independent import, in good agreement with the findings by Oliveira et al. [38]. Moreover, Pex5p was recycled multiple times between peroxisomes and the cytosol. Pex5p was efficiently exported from peroxisomes in the presence of an excess of Pex5p in the cytosol, implying that Pex5p is exported independently of its concentration gradient, consistent with the requirement for ATP.

Figure 2 A mechanistic model for matrix protein import, mediated by the shuttling receptor Pex5p and protein import machinery on peroxisomal membrane

Pex5p initially targets to the 800 kDa docking complex containing Pex14p and Pex13p, then translocates to the 500 kDa translocation complex composed of RING peroxins. At the terminal step of the protein import reaction, Pex1p and Pex6p catalyse the export of Pex5p. The numbers indicate peroxins. In the docking complex, (13) designates the possible involvement of Pex13p in the docking complex.

Pex1p and Pex6p are essential for Pex5p export, but not its import, consistent with the ATP-dependence of the export step. Organelle-associated Pex1p and Pex6p possibly play a major role in Pex5p export. Pex1p and Pex6p indeed bind to Pex5p [25]. Therefore it is conceivable that Pex1p and Pex6p may extract Pex5p from peroxisome membranes in an ATP-dependent manner. Such extraction activity is similar to that demonstrated by other members of the AAA family, such as p97, mitochondrial AAA proteases and bacterial FtsH, which are all tightly linked to substrate degradation [3941]. Distinct from these AAA proteins, Pex5p is destined for recycling instead of degradation. Such involvement of Pex1p and Pex6p in Pex5p relocation to the cytosol in S. cerevisiae was also reported recently [34].

Using Blue Native PAGE, we identified two novel and distinct Pex5p-containing import complexes in peroxisomal membranes, with molecular masses of approx. 800 and 500 kDa [25], as shown in Figure 2. The 800 kDa complexes contained Pex14p, and the 500 kDa one contained Pex2p. Pex14p has been shown to function as the initial docking site for Pex5p [15,35,42,43]. Thus the 800 kDa complexes are possibly the docking machinery for peroxisomal matrix proteins, and are likely to contain homo-oligomers of Pex14p [44]. Pex14p and Pex13p have been reported previously to be involved in the initial docking complexes of Pex5p [15,35,42,43]. Thus it is likely that Pex13p is present in the 800 kDa complexes. After targeting to peroxisomes via the 800 kDa complexes, Pex5p appears to translocate to the Pex2p-containing 500 kDa complexes. Three RING (really interesting new gene) peroxins, Pex2p, Pex10p and Pex12p, interact with each other and form RING core complexes [45,46]. Hence it is likely that the 500 kDa complexes also contain Pex10p and Pex12p. Consistent with this, in pex2 Z65 and pex12 ZP109 mutant cells, Pex5p translocated to the 800 kDa complexes, but not to the 500 kDa complexes [25]. Taken together, Pex5p targets Pex14p in the 800 kDa import machinery, then translocates to the 500 kDa complexes before finally exiting from peroxisomes in a process possibly mediated by the AAA peroxins.


The PEX26 gene, which is responsible for CG8 PBDs, encodes the C-terminally anchored Pex26p, the recruiter of Pex1p–Pex6p complexes to peroxisomes. ATP binding in both AAA cassettes, the D1 and D2 domains, but not ATP hydrolysis in the D2 domains of both Pex1p and Pex6p, was a prerequisite for Pex1p–Pex6p interaction and their localization in the peroxisome. Pex1p is found in two distinct oligomeric forms, a homo-oligomer in the cytosol and a hetero-oligomer on peroxisome membranes, and Pex1p possibly performs distinct functions in peroxisome biogenesis.

Pex5p is imported into and exported from peroxisomes for multiple cycles. ATP is not required for Pex5p import, but is indispensable for export. Pex1p and Pex6p of the AAA family and their recruiter Pex26p are essential for Pex5p export. Together, it is most likely that Pex5p enters peroxisomes, changes its interacting partners, then exits using energy from ATP. We await the delineation of further detailed mechanisms underlying peroxisomal protein import, including cargo unloading from Pex5p, as well as Pex5p export.


This work was supported in part by SORST (Solution Oriented Research for Science and Technology) and CREST (Core Research for Evolutional Science and Technology) grants from the Science and Technology Agency of Japan; Grants-in-Aid for Scientific Research; Grant of National Project on Protein Structural and Functional Analyses; and the 21st Century COE Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


  • 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; CG, complementation group; CHO, Chinese-hamster ovary; HEK-293, cells, human embryonic kidney cells; NSF, N-ethylmaleimide-sensitive fusion protein; PBD, peroxisome biogenesis disorder; PTS1, etc., peroxisome-targeting signal type 1 etc.; RING, really interesting new gene


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