CHARACTERIZATION OF A NOVEL COMPONENT OF THE PEROXISOMAL PROTEIN IMPORT APPARATUS USING FLUORESCENT PEROXISOMAL PROTEINS

Fluorescent peroxisomal probes were developed by fusing green fluoresc ent protein (GFP) to the matrix peroxisomal targeting signals PTS1 and PTS2, as well as to an integral peroxisomal membrane protein (IPMP). These proteins were used to identify and characterize novel peroxisome assembly (pas) mutants in the yeast Pichia pastoris. Mutant cells lac king the PAS10 gene mislocalized both PTS1-GFP and PTS2-GFP to the cyt oplasm but did incorporate IPMP-GFP into peroxisome membranes. Similar distributions were observed for endogenous peroxisomal matrix and mem brane proteins. While peroxisomes from translocation-competent pas mut ants sediment in sucrose gradients at the density of normal peroxisome s, >98% of peroxisomes from pas10 cells migrated to a much lower densi ty and had an extremely low ratio of matrix: membrane protein. These d ata indicate that Pas10p plays an important role in protein translocat ion across the peroxisome membrane. Consistent with this hypothesis, w e find that Pas10p is an integral protein of the peroxisome membrane. In addition, Pas10p contains a cytoplasmically-oriented C3HC4 zinc bin ding domain that is essential for its biological activity.