Interaction of Pex5p, the type 1 peroxisome targeting signal receptor, with the peroxisomal membrane proteins Pex14p and Pex13p

Pexp5p, a receptor for peroxisomal matrix proteins with a type 1 peroxisome targeting signal (PTS1), has been proposed to cycle from the cytoplasm to the peroxisomal membrane where it docks with Pex14p and Pex13p, the latter an SH3 domain-containing protein. Using in vitro binding assays we have dem onstrated that binding of Pex5p to Pex14p is enhanced when Pex5p is loaded with a PTS1-containing peptide. In contrast, Pex5p binding to Pex13p, which involves only the SH3 domain, occurs at 20-40-fold lower levels and is red uced when Pex5p is preloaded with a PTS1 peptide, Pex14p was also shown to bind weakly to the Pex13p SH3 domain. Site-directed mutagenesis of the Pex1 3p SH3 domain attenuated binding to Pex5p and Pex14p, consistent with both of these proteins being binding partners for this domain. The SH3 binding s ite in Pex5p was determined to lie within a 114-residue peptide (Trp(100)-G lu(213)) in the amino-terminal region of the protein. The interaction betwe en this peptide and the SH3 domain was competitively inhibited by Pex14p, W e interpret these data as suggesting that docking of the Pex5p-PTS1 protein complex at the peroxisome membrane occurs at Pex14p and that the Pex13p SH 3 domain functions as an associated component possibly involved in sequeste ring Pex5p after relinquishment of the PTS1 protein cargo to components of the translocation machinery.