PEROXISOME BIOGENESIS--A GENETIC APPROACH

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Eukaryotic cells possess a variety of organelles, each devoted to performing a specific set of metabolic functions. The cell must maintain each of these organelles and correctly direct specific sets of proteins to their proper subcellular locations. How the cell manages these operations for some organelles such as the mitochondrion, chloroplast and endoplasmic reticulum has been the subject of intensive investigations. Relative to these, little is known about peroxisomes, a family of ubiquitous organelles, surrounded by a single membrane and containing enzymes involved in a variety of important metabolic pathways. In humans, peroxisomal enzymes are particularly important in lipid metabolism (e.g., plasmalogen and bile acid synthesis; fatty acid, cholesterol and prostaglandin degradation) and genetic. defects in the organelles result in a lethal human disorder, termed Zellweger syndrome. The primary long-term goal of this program is to understand, at the molecular level, the mechanisms which control peroxisome biogenesis and function. The objective of the program is to initiate a genetic approach toward this end using the methanol-utilizing yeast Pichia pastoris as a model. This yeast was selected because peroxisomes are absolutely required for its growth on methanol, an easily observed phenotype, and because methods for classical- and molecular-genetic manipulation of the organism are fully developed. A comprehensive collection of peroxisome-deficient (per) mutants of P. pastoris will be isolated and subjected to genetic, biochemical and cellular biological studies aimed at elucidating the molecular basis of their primary defects. The per mutants will then be utilized to clone the specific genes affected and the DNA sequence of each PER gene will be determined and analyzed. The amino acid sequence deduced from each gene along with the biochemical analysis of the effect of mutations in each gene should help to elucidate the role of each protein in peroxisome function and to formulate an overall picture of peroxisomal operations. A second long-term goal of this program is to utilize knowledge gained from this research to understand the human disease state and to aid Zellweger victims, specifically. It is expected that some of the yeast peroxisomal genes that are identified will be homologues of genes that are affected in Zellweger patients. If so, information obtained on yeast PER genes and their products could be useful in identifying and isolating the human homologues. These human PER genes would be valuable as diagnostic probes and eventually may be used in gene therapy treatments.