- Top of page
- Materials and Methods
A peroxisomal C-tail-anchored type-II membrane protein, Pex26p, recruits AAA ATPase Pex1p–Pex6p complexes to peroxisomes. We herein attempted to gain mechanistic insight into Pex26p function. Pex26pΔ33-40 truncated in amino-acid residues at 33-40 abolishes the recruiting of Pex1p–Pex6p complex to peroxisomes and fails to complement the impaired phenotype of pex26 CHO cell mutant ZP167, thereby suggesting that peroxisomal localization of Pex1p and Pex6p is indispensable for the transport of matrix proteins. In in vitro transport assay using semipermeabilized CHO cells, Pex1p is targeted to peroxisomes in a manner dependent on ATP hydrolysis, while Pex6p targeting requires ATP but not its hydrolysis. This finding is confirmed by the assay using Walker-motif mutants. Transport of Pex1p and Pex6p is temperature-dependent. In vitro binding assays with glutathione-S-transferase-fused Pex26p, Pex1p and Pex6p bind to Pex26p in a manner dependent on ATP binding but not ATP hydrolysis. These results suggest that ATP hydrolysis is required for stable localization of Pex1p to peroxisomes, but not for binding to Pex26p. Moreover, Pex1p and Pex6p are altered to a more compact conformation upon binding to ATP, as verified by limited proteolysis. Taken together, Pex1p and Pex6p are most likely regulated in their peroxisomal localization onto Pex26p via conformational changes by the ATPase cycle.
Peroxisome, a single membrane-bounded ubiquitous organelle, is present in a wide variety of eukaryotic cells from yeast to humans, and functions in various metabolic pathways, including β-oxidation of very long-chain fatty acids and the synthesis of ether lipids (1).
Up to now, we and other groups have revealed 14 different complementation groups (CGs) in peroxisome-deficient phenotypes in mammals, by CG analysis between peroxisome-deficient CHO cell mutants and fibroblasts from patients with peroxisome biogenesis disorders (PBDs). Therefore, at least 14 genes (PEX) or their products, termed peroxins, are required for peroxisome biogenesis in mammals (2–6). Import of peroxisomal matrix proteins is mediated by two types of peroxisomal-targeting signals (PTSs): PTS1, identified in numerous enzymes (7–9), and PTS2, present in several proteins such as 3-ketoacyl-CoA thiolase (thiolase) (10,11). In the cytoplasm, Pex5p and Pex7p function as the PTS1 and PTS2 receptors, respectively, and translocate respective PTS-cargoes into peroxisomes via peroxisomal membrane importers (12–15). In contrast to the molecular mechanisms underlying the matrix protein import to peroxisomes, those of peroxisome membrane biogenesis are less understood (16–18).
PEX26 is identified the most recently identified gene responsible for PBDs of 13 CGs (2,19). PEX26 encodes a 34-kDa type-II peroxisomal membrane protein with one C-terminal transmembrane segment, exposing its N-terminal domain to the cytosol (2). Pex26p interacts with Pex1p–Pex6p complexes and recruits them to peroxisomes (2,20–22). Yeast Pex15p, a functional homolog of mammalian Pex26p, binds Pex6p (23) and forms a complex with Pex1p and Pex6p (24). Pex1p and Pex6p are members of the AAA ATPase family (5,25–29) and show a dual subcellular localization, mostly in peroxisomes and partly in the cytosol (21,24,30,31). Pex1p and Pex6p are required for the export of Pex5p from peroxisomes. However, it remains unclear how the AAA peroxins export Pex5p to the cytosol. Furthermore, ATP requirements for the Pex1p–Pex6p complex and targeting mechanism of Pex1p and Pex6p to peroxisomes remain obscure.
As a further step toward understanding the molecular mechanisms underlying the targeting of Pex1p and Pex6p to Pex26p and its regulation, we here developed an in vitro assay system for targeting of Pex1p and Pex6p to Pex26p using semi-intact cells. Moreover, we provide several lines of evidence that peroxisomal targeting of Pex1p and Pex6p takes place in an ATP-dependent manner and that nucleotide binding to Pex1p and Pex6p triggers their conformational changes.
- Top of page
- Materials and Methods
In the present work, we identified the region of Pex26p involved in the peroxisomal targeting of Pex1p and Pex6p. The N-terminal cytoplasmic domain of the integral peroxisomal membrane protein Pex26p is required for peroxisomal targeting of Pex1p–Pex6p complexes and peroxisomal matrix protein import (2,20,22) (Figure 1). Similarly in the yeast Saccharomyces cerevisiae, Pex15p functions as the homolog of mammalian Pex26p (23). Pex6p interacts with the cytoplasmic part of Pex15p and the assembly of Pex1p–Pex6p–Px15p complexes is crucial for matrix protein import (23,24). Importantly, Pex26pΔ33-40 with a deletion of eight amino acids at 33-40 is compromised in the activity in recruiting Pex1p and Pex6p, thereby giving rise to the defect of matrix protein import into peroxisomes. These results raise several possibilities with respect to the relationship between the function and peroxisomal localization of these two AAA peroxins. One possibility is that peroxisomal localization of Pex1p and Pex6p is indispensable for the transport of matrix proteins including catalase and PTS1 and PTS2 proteins. However, several PBD patient-derived Pex26p mutants showing insufficient binding to Pex1p and Pex6p, hence indicative of cytosolic localization of the Pex1p–Pex6p complexes, were partially competent in the import of PTS1, but less efficient for the import of catalase and PTS2 proteins (19,20,22). Therefore, a part of Pex1p and Pex6p in the cytosol might be responsible for PTS1 protein import. Nevertheless, peroxisome-associated Pex1p and Pex6p are required for the efficient import of PTS1 proteins. It is also plausible that N-terminal domain of Pex26p functions with auxiliary factors besides Pex1p–Pex6p complexes in the transport of matrix proteins. PEX26 mutations identified in the PBD patients of CG8 are indeed located mostly at the genome sequence encoding the N-terminal domain of Pex26p (19,20,22), implying that this portion is crucial for the Pex26p function.
By making use of the semi-intact cell assay system successfully established in the present work, we showed that the peroxisomal targeting of Pex1p and Pex6p was enhanced by heat-labile factor(s) from the cytosolic fraction, implying that protein factors are involved in this process. To further characterize such factors, we performed immunodepletion assays with anti-HSP70 antibody. HSP70 depletion did not affect the peroxisomal targeting of Pex1p and Pex6p. We also showed that heat-inducible proteins, including HSP70 and HSP40, did not enhance the peroxisomal targeting of Pex1p and Pex6p (Figure 3). Any potential cytosolic protein(s) remain to be defined.
Limited digestion with trypsin has been used to verify the conformational changes of a number of AAA proteins such as NEM-sensitive factor (NSF) (34). On the basis of the patterns of limited tryptic digests of Pex1p or Pex6p under different nucleotide conditions (Figure 7), we conclude that conformational changes of Pex1p and Pex6p are induced upon binding to ATP and a little with AMP-PNP (Figure 7). In the absence of nucleotides, Pex1p and Pex6p show relatively loose and trypsin-sensitive conformation. Upon binding to nucleotides, Pex1p and Pex6p undergo a conformational change, becoming rather resistant to trypsin. It is also noteworthy that Pex1p fragments with lower molecular masses, apparently below ∼50 kDa, were detected only with the antiserum specific to the N-terminal, not C-terminal, part of Pex1p (21,25), thereby suggesting that the C-terminal part of Pex1p represents a loose and flexible conformation sensitive to trypsin. Intriguingly, ∼30-kDa fragment (Figure 7A, left panels, downward open arrowhead) is more resistant to tryptic cleavage in the presence of ATP than in the absence of any externally added nucleotides or in the presence of AMP-PNP. While the ∼30-kDa fragment of Pex1p appears to be further degraded to a 25-kDa fragment, both detectable with anti-N-terminal domain antibody, it is conceivable that an ATP hydrolysis-driven structural change gives rise to the upper fragment band protected from the tryptic digestion. The N-terminal domain of Pex1p is suggested to bind phosphoinositides and thereby associate with the peroxisomal membrane by recognizing specific lipids (39). Pex1p binding to the peroxisomal membrane may be regulated by an N-terminal conformational change dependent on the nucleotide-bound state.
With the semi-intact cell assay system, we show that the peroxisomal targeting of Pex1p requires ATP hydrolysis (Figure 4). In GST pull-down assays, Pex1p binds to Pex26p in a manner dependent on Pex6p and ATP binding, but not requiring ATP hydrolysis (Figure 6). We interpret this difference in regard to the requirement of ATP hydrolysis to imply that the interactions between Pex1p and phospholipids on the peroxisomal membrane surface are also involved in the regulation of Pex1p targeting. Another possibility is that Pex1p-interacting proteins including membrane-associated proteins and/or soluble peroxins such as Pex5p influence the Pex1p complex formation. On the other hand, Pex6p interacts with Pex26p in a manner dependent on ATP binding, but not requiring ATP hydrolysis, both in semi-intact cell assays and in GST pull-down assays (Figures 4 and 6). Therefore, it is more likely that Pex1p and Pex6p translocate to peroxisomes in a mutually distinct manner. Similar results were obtained in semi-intact cell assays using Walker-motif mutants of Pex1p and Pex6p, where the Walker A mutants influencing the ATP binding were more severely affected in targeting to Pex26p than the Walker B mutants (Figure 5). Pex6p mutated in the ATP-binding site (A1 or A2) shows a reduced level of targeting to Pex26p, while a point mutation in the ATP-hydrolysis site (B2) does not affect the peroxisomal targeting of Pex6p. These results suggest that the conformational changes in both D1 and D2 domains of Pex6p upon ATP binding, not its hydrolysis, are critical for the translocation of Pex6p to peroxisomes. With regard to Pex1p, the Walker mutants, A1, B1, and A2, show weaker interaction with Pex6p–Pex26p complexes on peroxisomes as compared to the wild type, while Pex1pB2 is transported to peroxisomes as efficiently as the wild-type Pex1p (Figure 5). Accordingly, it is more likely that ATP binding in both AAA cassettes and the following ATP hydrolysis in D1 of Pex1p are prerequisites for peroxisomal localization (Figure 8).
Figure 8. A model for peroxisomal targeting of Pex1p and Pex6p. A schema of conformational changes in the AAA ATPases, Pex1p and Pex6p (represented as homo-hexamers), between three different states: ATP-bound, ATP-hydrolyzing and nucleotide-free conditions. Stages: (i) in the absence of ATP, Pex1p and Pex6p show relatively ‘less compact' structure; (ii) upon binding to ATP, their conformations become more compact; (iii) at the following ATP-hydrolyzing step, both peroxins alter to a closed and tight configuration. Pex1p and Pex6p in the stage (iii) are translocated to peroxisomes. Pex6p in the stage (ii) is also competent for targeting to peroxisomes.
Download figure to PowerPoint
Crystal structure of the N-terminal domain of mouse Pex1p resembles valocin-containing protein (VCP), another member of the AAA proteins (39,40). VCP forms a barrel-like homo-hexameric structure (41). Recently, we showed that Pex1p is in a homo-trimer and a homo-hexamer (21). VCP as well as Pex1p possesses a highly protease-sensitive C-terminal domain, which is conversely protected from tryptic digestion upon nucleotide binding in the case of VCP (36). Thus, it is plausible that Pex1p forms a hexameric ring structure upon ATP binding and undergoes a structural change during the ATPase cycle in a manner similar to VCP (Figure 8). Likewise, ATP binding to Pex6p may trigger a conformational change and regulate its homo-oligomerization.
Pex1p and Pex6p are involved in early and late steps of peroxisome biogenesis including membrane fusion (42) and protein dislocation such as Pex5p export from peroxisomes (12,13,43). The yeast Pex1p and Pex6p likewise play pivotal roles in peroxisome biogenesis (13,17,23,24). These functions of Pex1p and Pex6p are reminiscent of those of NSF and VCP. AAA ATPases play various roles including vesicle fusion by NSF (44,45) and endoplasmic reticulum (ER)-associated protein degradation by p97/VCP/CDC48 (46). Despite their main localization in the cytosol, NSF and p97 exert their functions on membranes in vesicle fusion (47,48) and ER-associated protein degradation (49), respectively. AAA ATPase peroxins, Pex1p and Pex6p, also function on peroxisomal membranes to export Pex5p to the cytosol for a next round of protein import. Therefore, it is a prerequisite for AAA ATPases to target organelle membranes where they function. In the present work, we have dissected the mechanisms underlying the targeting of AAA ATPase peroxins to peroxisomes. As shown in Figure 8, our findings strongly suggest that ATP-dependent binding of Pex1p and Pex6p to and possibly their release from Pex26p via concomitant conformational changes are responsible for the regulation of subcellular localization and functions of AAA ATPase peroxins. Furthermore, our newly developed transport system using semi-intact cells readily analyzes the dynamics of Pex1p and Pex6p targeting to and release from their recruiter Pex26p under the physiological conditions. This system would open a way to elucidate the mechanisms underlying peroxisome biogenesis involving matrix protein import.