ABSTRACT
Peroxisomal proteins carrying a peroxisome targeting signal type 1 (PTS1) are recognized in the cytosol by the cycling import receptor Pex5p. The receptor-cargo complex docks at the peroxisomal membrane where it associates with multimeric protein complexes, referred to as the docking and RING finger complexes. Here we have identified regions within the Saccharomyces cerevisiae Pex5p sequence that interconnect the receptor-cargo complex with the docking complex. Site-directed mutagenesis of the conserved tryptophan residue within a reverse WXXXF motif abolished two-hybrid binding with the N-terminal half of Pex14p. In combination with an additional mutation introduced into the Pex13p-binding site, we generated a Pex5p mutant defective in a stable association not only with the docking complex but also with the RING finger peroxins at the membrane. Surprisingly, PTS1 proteins are still imported into peroxisomes in these mutant cells. Because these mutations had no significant effect on the membrane binding properties of Pex5p, we examined yeast and human Pex5p for intrinsic lipid binding activity. In vitro analyses demonstrated that both proteins have the potential to insert spontaneously into phospholipid membranes. Altogether, these data strongly suggest that a translocation-competent state of the PTS1 receptor enters the membrane via protein-lipid interactions before it tightly associates with other peroxins.