Peroxisomes are present in cells of virtually all eukaryotes and are the subcellular location of many important metabolic reactions. Little is known regarding basic mechanisms by which cells maintain and propagate peroxisomes and direct specific sets of newly synthesized proteins to these organelles. This laboratory has initiated a molecular genetic investigation of peroxisomes using the methylotrophic yeast, Pichia pastoris, as a model system. P. pastoris was chosen because peroxisomes are required for its growth on either methanol or oleate (an easily observed phenotype) and because molecular genetic methods are well developed in this organism. By isolating and examining a collection of methanol- utilization-defective P. pastoris strains, 23 mutants in 8 different genes have been identified that are peroxisome-deficient (per mutants). These mutants will be subjected to genetic, biochemical and cellular biological studies to elucidate their primary defects. Selected mutants will also be used to clone PER genes by functional complementation. Information from predicted amino acid sequences of PER products together with that from biochemical analysis of per mutants should yield important insights into the function of specific PER proteins and into the mechanisms that operate in peroxisome biogenesis. %%% Virtually all eukaryotic cells harbor membrane-bound organelles called peroxisomes. They are the site of hydrogen peroxide- generating oxidative reactions in cells. Peroxisomal matrix proteins catalyze several important metabolic pathways; the specific pathways vary depending on the organism and/or organ or tissue. In plants, peroxisomes are involved in photorespiration and, during germination of fatty seeds, contain enzymes responsible for the beta-oxidation of stored fatty acids to acetyl-CoA, which enters the glyoxylate cycle to form succinate, the metabolic building block. Glyoxylate cycle reactions may even occur in the peroxisome, in which case the organelle is called a glyoxysome. In animals, peroxisomal enzymes play an essential role in several anabolic and catabolic pathways, particularly in lipid metabolism; these include synthesis of plasmalogens (ether-linked membrane glycerolipids), oxidative degradation of very long chain fatty acids, and certain aspects of sterol and bile acid metabolism. A group of human genetic disorders, invariably fatal in infancy, are due to the absence of peroxisomes. Despite the universal importance of peroxisomes for eukaryotic organisms, very little is known about their biogenesis. They are not part of the exocytic / endocytic system of the cell (including lysosomes, endoplasmic reticulum, and Golgi apparatus), and they are also distinctly different from mitochondria and chloroplasts, which have double membrane boundaries and endogenous genomes and protein synthetic machineries. The P. pastoris system, developed in this laboratory, offers the opportunity of approaching the peroxisomal biogenesis problem from several different angles at once (biochemistry, genetics, and cell biology). This work will shed important new light on how peroxisomes are assembled in eukaryotic cells.
