Peroxisomes play a role in lipid metabolism. At least 25 human diseases affect the function or subcellular location of peroxisomal enzymes, or impair peroxisome biogenesis. The targeting of proteins to the peroxisomal matrix and membrane is mediated by peroxisomal targeting signals (PTSs), of which PTS1 and PTS2 allow import of proteins to the matrix, and the mPTS facilitates import to the membrane. Two key peroxins involved in matrix protein import, Pex5p and Pex7p, bind the PTS1 and PTS2 sequences, respectively, in the cytosol. The resulting cargo-receptor complexes dock with other peroxins on the peroxisomal membrane, before cargo translocation occurs into the organelle matrix. The different results obtained regarding the subcellular locations of Pex5p and Pex7p can be reconciled by the hypothesis that PTS receptors shuttle from the cytosol to the peroxisome matrix and then return to the cytosol during the import cycle. We have provided evidence, since the last submission, for this extended-shuttle model for human Pex5p (HsPex5p). Our proposed work focuses on the itineraries of Pex5p and Pex7p during the import cycle in human and yeast cells, and the mechanistic details of various steps of this shuttling cycle. Our studies will reveal the mechanism of action of these receptors, which are mutated in fatal disorders and are unique relative to signal-sequence receptors used by other organelles. The aims are:1. What are the segments of HsPex5p that allow its import and export into and out of peroxisomes? Do different isoforms of HsPex5p shuttle? Are cargo binding or interactions of HsPex5p with the peroxisomal docking and translocation machinery necessary for its shuttling?2. Is the shuttling of Pex5p into and out of peroxisomes conserved in yeasts?3. What is the mechanism of steps in Pex5p shuttling such as transfer to the translocon, membrane translocation, cargo release and receptor export? We will also reconstitute receptor shuttling in vitro.4. What peroxins are necessary for the shuttling of Pex5p into and out of peroxisomes?5. Does Pex7p cycle into and out of the peroxisome matrix in yeast and in mammals? If so, what is the mechanism by which Pex7p shuttles?