Insertion of Pex5p into the Peroxisomal Membrane Is Cargo Protein-dependent

Abstract

It is now generally accepted that Pex5p, the receptor for most peroxisomal matrix proteins, cycles between the cytosol and the peroxisomal compartment. According to current models of peroxisomal biogenesis, this intracellular trafficking of Pex5p is coupled to the transport of newly synthesized peroxisomal proteins into the organelle matrix. However, direct evidence supporting this hypothesis was never provided. Here, using an in vitro peroxisomal import system, we show that insertion of Pex5p into the peroxisomal membrane requires the presence of cargo proteins. Strikingly the peroxisomal docking/translocation machinery is also able to catalyze the membrane insertion of a Pex5p truncated molecule lacking any known cargo-binding domain. These results suggest that the cytosol/peroxisomal cycle in which Pex5p is involved is directly or indirectly regulated by Pex5p itself and not by the peroxisomal docking/translocation machinery.

A Novel Pex14 Protein-interacting Site of Human Pex5 Is Critical for Matrix Protein Import into Peroxisomes

“…Dissociation and sliding along the Pex5 polypeptide chain could initiate further binding of Pex14 to WXXX(F/Y) motifs 1-5 resulting in the formation of a multimeric Pex5-Pex14 complex that presumably represents the protein-conducting channel enabling matrix protein translocation across the peroxisomal membrane. Interestingly, a stable subcomplex consisting of Pex5 and Pex14 with a 1:5 stoichiometry could be isolated from rat liver peroxisomes (6). Finally, Pex14 binding to the downstream-located WXXX(F/Y) motifs six and seven triggers cargo release (10).…”

“…The PTS1 motif interacts with the tetratricopeptide repeat domain in the C-terminal half of Pex5. The intrinsically disordered N-terminal half of Pex5 per se is capable of performing all transport steps of the receptor cycle, including docking and pore formation and dislocation from the peroxisomal membrane (5,6).…”

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

“…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).…”

“…40), GST-Pex5p (39), GST-SKL (GST containing a PTS1 signal at the C terminus; Ref. 40), and GST-LKS (GST ending with a nonfunctional PTS1-like sequence; Ref. 40) were described before.…”

“…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.…”