Characterization of the Peroxisomal Cycling Receptor, Pex5p, Using a Cell-free in Vitro Import System

Abstract

According to current models of peroxisomal biogenesis, Pex5p cycles between the cytosol and the peroxisome transporting newly synthesized proteins to the organelle matrix. However, little is known regarding the mechanism of this pathway. Here, we show that Pex5p enters and exits the peroxisomal compartment in a process that requires ATP. Insertion of Pex5p into the peroxisomal membrane is blocked by anti-Pex14p IgGs. At the peroxisomal level, two Pex14p-associated populations of Pex5p could be resolved, stage 2 and stage 3 Pex5p, both exposing the majority of their masses into the organelle lumen. Stage 3 Pex5p can be easily detected only under ATP-limiting conditions; in the presence of ATP it leaves the peroxisomal compartment rapidly. Our data suggest that translocation of PTS1-containing proteins across the peroxisomal membrane occurs concomitantly with formation of the Pex5p-Pex14p membrane complex and that this is probably the site from which Pex5p leaves the peroxisomal compartment.

The N Terminus of the Peroxisomal Cycling Receptor, Pex5p, Is Required for Redirecting the Peroxisome-associated Peroxin Back to the Cytosol

“…1A, lane 6), it is no longer possible to detect two protease-resistant populations. Please note that the difference between stage 2 and stage 3 Pex5p is that only the former can be cleaved by proteinase K, a cleavage that occurs around amino acid residues 15-20 (39). Obviously, in the absence of this domain, as it is the case for ⌬N110-Pex5p, no cleavage can occur.…”

“…11) or the C-terminal truncated version comprising amino acid residues 1-324 of Pex5p (⌬C1-Pex5p) preceded by the T7 RNA polymerase promotor were obtained as described previously (39,40). The cDNAs encoding the N-terminal truncated versions of Pex5p lacking the first 17 or the first 110 amino acid residues (⌬N17-Pex5p and ⌬N110-Pex5p, respectively) were obtained by using an expression PCR strategy (41).…”

“…With this modification the Pex5p export activity of peroxisomes is maintained for longer periods (ϳ45 min; data not shown). Proteinase K treatment of import reactions and processing of protein samples for SDS-PAGE and autoradiography were done exactly as described (39).…”

“…In contrast, export (recycling) of Pex5p from the peroxisomal compartment is ATP-dependent (34). At the peroxisomal membrane level, two different populations of Pex5p could be identified and characterized using protease-protection assays (39). In the presence of ATP, conditions in which Pex5p continuously enters and exits the peroxisomal membrane, peroxisomal Pex5p behaves as a transmembrane protein exposing the majority of its mass into the peroxisomal lumen with only a small N-terminal fragment of ϳ2 kDa accessible to the protease from the cytosolic side of the membrane.…”

“…It consists of incubating 35 S-labeled Pex5p with a post-nuclear supernatant from rat liver, and it explores the fact that Pex5p acquires a protease-resistant status when inserted into the peroxisomal membrane (38,39). By using this experimental strategy, it was shown that insertion of Pex5p into the peroxisomal membrane requires the presence of PTS1-containing proteins in the import medium and depends on available Pex14p, an intrinsic membrane component of the peroxisomal docking/translocation machinery (39,40). Unexpectedly, insertion of Pex5p into the organelle membrane does not require ATP, suggesting that the driving force for protein translocation across the peroxisomal membrane resides on protein-protein interactions.…”