Interaction of Pex5p, the type 1 peroxisome targeting signal receptor, with the peroxisomal membrane proteins Pex14p and Pex13p.
Pex5p, 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 demonstrated 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 reduced 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 Pex13p SH3 domain attenuated binding to Pex5p and Pex14p, consistent with both of these proteins being binding partners for this domain. The SH3 binding site in Pex5p was determined to lie within a 114-residue peptide (Trp(100)-Glu(213)) in the amino-terminal region of the protein. The interaction between this peptide and the SH3 domain was competitively inhibited by Pex14p. We interpret these data as suggesting that docking of the Pex5p-PTS1 protein complex at the peroxisome membrane occurs at Pex14p and that the Pex13p SH3 domain functions as an associated component possibly involved in sequestering Pex5p after relinquishment of the PTS1 protein cargo to components of the translocation machinery.
Urquhart
A J
AJ
School of Biomolecular and Biomedical Science, Griffith University, Nathan, Queensland 4111, Australia.
Kennedy
D
D
Gould
S J
SJ
Crane
D I
DI
D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins; D005656:Fungal Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D000074401:Peroxins; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000378:metabolism*; D010843:Pichia; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D018909:src Homology Domains / Q000235:genetics
D002352:Carrier Proteins; D005656:Fungal Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C101884:PEX13 protein, human; C499195:PEX14 protein, S cerevisiae; D000074401:Peroxins; D000074435:Peroxisome-Targeting Signal 1 Receptor; D021382:Protein Sorting Signals; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins