pas mutants of Saccharomyces cerevisiae are disturbed in peroxisome assembly (pas) and proliferation. Here we report the characterization of the PAS10 gene and its product (PAS10) that is essential for the import of a large subset of proteins into the peroxisomal matrix. PAS10, a protein of 69 kDa, is a member of the tetratricopeptide repeat, or snap helix, protein family, characterized by several direct repeats of a degenerate 34-amino acid motif (Sikorski, R. S., Boguski, M. S., Goebl, M. & Hieter, P. (1990) Cell 60, 307-317). Other members of this family are MAS70 (S. cerevisiae) and MOM72 (Neurospora crassa), which are mitochondrial receptors for protein import. A pas10 null mutant accumulates peroxisomal, leaflet-like membrane structures and exhibits deficient import of a number of peroxisomal matrix enzymes, particularly of proteins with an SKL-like import signal. In contrast, 3-ketoacyl-CoA thiolase associated with these membranes is resistant in vitro to degradation by proteinase K, indicating true protein import. These results suggest that PAS10 is an essential component of a peroxisomal import machinery which mediates the translocation of a specific subset of proteins to the peroxisomal matrix with an SKL-like import signal.

Abstract. We have developed a positive selection system for the isolation of Saccharomyces cerevisiae mutants with disturbed peroxisomal functions. The selection is based on the lethality of hydrogen peroxide (H202) that is produced in wild type cells during the peroxisomal/~-oxidation of fatty acids. In total, 17 mutants having a general impairment of peroxisome biogenesis were isolated, as revealed by their inability to grow on oleic acid as the sole carbon source and their aberrant cell fractionation pattern of peroxisomal enzymes. The mutants were shown to have monogenetic defects and to fall into 12 complementation groups. Representative members of each complementation group were morphologically examined by immunocytochemistry using EM. In one mutant the induction and morphology of peroxisomes is normal but import of thiolase is abrogated, while in another the morphology differs from the wild type: stacked peroxisomal membranes are present that are able to import thiolase but not catalase. These mutants suggest the existence of multiple components involved in peroxisomal protein import. Some mutants show the phenotype characteristic of glucose-repressed cells, an indication for the interruption of a signal transduction pathway resulting in organelle proliferation. In the remaining mutants morphologically detectable peroxisomes are absent: this phenotype is also known from fibroblasts of patients suffering from Zellweger syndrome, a disorder resulting from impairment of peroxisomes.

We report the characterization of ScPex8p, which is essential for peroxisomal biogenesis in Saccharomyces cerevisiae. Cells lacking Pex8p are characterized by the presence of peroxisomal membrane ghosts and mislocalization of peroxisomal matrix proteins of the PTS1 and PTS2 variety to the cytosol. Pex8p is tightly associated with the lumenal face of the peroxisomal membrane. Consistent with its intraperoxisomal localization, Pex8p contains a peroxisomal targeting signal 1, and it interacts with the PTS1 receptor Pex5p. However, the Pex5p/ Pex8p association is also observed upon deletion of the PTS1 of Pex8p, suggesting that Pex8p contains a second binding site for Pex5p. The pex8⌬ mutant phenotype and the observed PTS1-independent interaction with the PTS1 receptor suggest that Pex8p is involved in protein import into the peroxisomal matrix. In pex8⌬ cells, the PTS1 and PTS2 receptor still associate with membrane bound components of the protein import machinery, supporting the assumption that the Pex8p function in protein translocation follows the docking event.

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