A MOBILE PTS2 RECEPTOR FOR PEROXISOMAL PROTEIN IMPORT IN PICHIA-PASTORIS

Using a new screening procedure for the isolation of peroxisomal impor t mutants in Pichia pastel-is we have isolated a mutant (pex7) that is specifically disturbed in the peroxisomal import of proteins containi ng a peroxisomal targeting signal type II (PTS2). Like its Saccharomyc es cerevisiae homologue, PpPex7p interacted with the PTS2 in the two-h ybrid system, suggesting that Pex7p functions as a receptor, The pex7 Delta mutant was not impaired for growth on methanol, indicating that there are no PTS2-containing enzymes involved in peroxisomal methanol metabolism. In contrast, pex7 Delta cells failed to grow on oleate, bu t growth on oleate could be partially restored by expressing thiolase (a PTS2-containing enzyme) fused to the PTS1. Because the subcellular location and mechanism of action of this protein are controversial, we used various methods to demonstrate that Pex7p is both cytosolic and intraperoxisomal. This suggests that Pex7p functions as a mobile recep tor, shuttling PTS2-containing proteins from the cytosol to the peroxi somes, In addition, we used PpPex7p as a model protein to understand t he effect of the Pex7p mutations found in human patients with rhizomel ic chondrodysplasia punctata, The corresponding PpPex7p mutant protein s were stably expressed in P. pastoris, but they failed to complement the pex7 Delta mutant and were impaired in binding to the PTS2 sequenc e.