Saccharomyces cerevisiaePTS1 Receptor Pex5p Interacts with the SH3 Domain of the Peroxisomal Membrane Protein Pex13p in an Unconventional, Non-PXXP–related Manner

Abstract

A number of peroxisome-associated proteins have been described that are involved in the import of proteins into peroxisomes, among which is the receptor for peroxisomal targeting signal 1 (PTS1) proteins Pex5p, the integral membrane protein Pex13p, which contains an Src homology 3 (SH3) domain, and the peripheral membrane protein Pex14p. In the yeast Saccharomyces cerevisiae, both Pex5p and Pex14p are able to bind Pex13p via its SH3 domain. Pex14p contains the classical SH3 binding motif PXXP, whereas this sequence is absent in Pex5p. Mutation of the conserved tryptophan in the PXXP binding pocket of Pex13-SH3 abolished interaction with Pex14p, but did not affect interaction with Pex5p, suggesting that Pex14p is the classical SH3 domain ligand and that Pex5p binds the SH3 domain in an alternative way. To identify the SH3 binding site in Pex5p, we screened a randomly mutagenized PEX5 library for loss of interaction with Pex13-SH3. Such mutations were all located in a small region in the N-terminal half of Pex5p. One of the altered residues (F208) was part of the sequence W204XXQF208, that is conserved between Pex5 proteins of different species. Site-directed mutagenesis of Trp204 confirmed the essential role of this motif in recognition of the SH3 domain. The Pex5p mutants could only partially restore PTS1-protein import in pex5Δ cells in vivo. In vitro binding studies showed that these Pex5p mutants failed to interact with Pex13-SH3 in the absence of Pex14p, but regained their ability to bind in the presence of Pex14p, suggesting the formation of a heterotrimeric complex consisting of Pex5p, Pex14p, and Pex13-SH3. In vivo, these Pex5p mutants, like wild-type Pex5p, were still found to be associated with peroxisomes. Taken together, this indicates that in the absence of Pex13-SH3 interaction, other protein(s) is able to bind Pex5p at the peroxisome; Pex14p is a likely candidate for this function.

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

“…Prominent examples are two typical WXXX(F/Y) motifs of S. cerevisiae Pex5 (18,21) or one of the three WXXX(F/Y) motifs of Trypanosoma brucei Pex5 (22). These sequences may mediate the interaction with other peroxins, as shown for human and yeast Pex5 motifs, which both can interact with the Src homology 3 domain of Pex13 (8,13,24,25).…”

“…A peroxisomal membrane complex consisting of Pex13 and Pex14 (and Pex17 in yeast) provides the primary docking site for the cargo-loaded receptors (7,8). Pex14 fulfills additional important functions in cargo translocation across the peroxisomal membrane.…”

Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes

“…In mammals, GST (glutathione transferase)-PEX13 pull-down assays have shown that the N-terminal 100 amino acids provide a binding domain for PEX5 (Otera et al, 2002), whereas in yeast, pull-down and two-hybrid assays have shown that the C-terminal SH3 (Src homology 3) domain provides a binding site for Pex5p (Bottger et al, 2000;Urquhart et al, 2000). A. thaliana PEX7 also bind the N-terminus of PEX13 in a yeast two-hybrid assay (Mano et al, 2006).…”

Peroxisomal Targeting Signal Receptor Pex5p Interacts with Cargoes and Import Machinery Components in a Spatiotemporally Differentiated Manner: Conserved Pex5p WXXXF/Y Motifs Are Critical for Matrix Protein Import

“…In S. cerevisiae, the pentapeptide motifs have been demonstrated to be involved in the interaction of Pex5p with Pex13p (5). However, mutation of the motifs did not affect the binding of Pex5p with Pex14p, implying that the WXXXF/Y motifs are not involved in such an interaction (5). Whether or not the WXXXF/Y motifs are involved in the interaction of mammalian Pex5p with Pex13p has not been reported, but seven pentapeptide motifs in human Pex5pL have been shown to bind Pex14p in vitro with different affinities (35).…”

“…Pex5p is composed of two distinct parts, a highly conserved C-terminal half comprising seven tetratricopeptide repeat (TPR) motifs and an N-terminal half in which only a few amino acids are strictly conserved, typically in the multiple pentapeptide WXXXF/Y repeats (5,36). While the TPR region was shown to mediate the binding to PTS1-containing proteins (18), specific functions were not fully assigned to the N-terminal half.…”