Peroxisomes are subcellular organelles found in all eukaryotic cells. They are simple in composition, a single membrane surrounds a small number of matrix proteins, contain no DNA and have different functions in different cell types, a unique property amongst organelles. In liver cells, peroxisomes are responsible for beta-oxidation of long-chain fatty acids and for plasmalogen biosynthesis. There exist several devastating hereditary human disease states caused by a lack of one or more peroxisomal functions. Zellweger's cerebrohepatorenal syndrome is characterized by an almost complete absence of kidney and liver peroxisomes, and it has been proposed that the underlying biochemical fault in this disease is an inability to assemble peroxisomes. Unfortunately, the mechanisms of assembly of peroxisomes are unknown at present, and the long-term objectives of this research are to elucidate the mechanisms involved in that process. Yeast of the Candida and Hansenula genera elaborate large quantities of peroxisomes when grown on methanol and thus provide a convenient model system for these studies.
The specific aims of the current proposal are to determine mechanisms of import into peroxisomes of cytoplasmically synthesized alcohol oxidase, the major peroxisomal matrix protein in these yeasts. Work will be done using certain mutant strains, unable to grow on methanol, that have the extremely useful property of being able to synthesize alcohol oxidase after exposure to methanol at 37 degrees but not at 21 degrees. It is believed that at 21 degrees these strains are unable to assemble competent peroxisomes and thus may serve as excellent model systems for Zellweger's disease and its study. An investigation into the exact nature of the defect in these mutants could increase knowledge about the fundamental defect in the disease state, and also contribute basic understanding of peroxisome assembly in general. Methodologies to be used to study the mutants include: (1) pulse labelling experiments with (35S) methionine of spheroplasts after different conditions of growth to reveal the mechanism of processing of alcohol oxidase and its relationship to peroxisome formation, (2) electron microscopy studies to determine what enzymes they contain, and (3) chemical studies on the composition of mutant peroxisomal membranes. Additionally, attempts to obtain a viable in vitro import system for isolated peroxisomes will be undertaken. This will be done by purifying peroxisomes from different cell types and determining their capability for import of in vitro synthesized proteins using octamerization of alcohol oxidase, identified by immunoprecipitation, as the criterion. If successful, this system will be sued to probe the involvement in the import system of energy requirements, the presence of specific proteinaceous receptors on the peroxisomal membrane and the role of coenzyme attachment in the process.
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