The fundamental mechanisms of sorting of proteins and assembly of organelles are still largely unresolved. In particular, the biogenesis of peroxisomes, essential and ubiquitous organelles, has been little studied. Defects in peroxisomal assembly can lead to catastrophic human disease. The methanol-induced yeast peroxisome, simple in structure and function, abundant and highly inducible, is a promising model to study basic questions of organellar biogenesis in general and peroxisomal assembly in particular. The long range goal of this work is to understand the interactions of sorting signals on peroxisomal proteins with the membrane to cause import. Two approaches will be undertaken to elucidate assembly of the methanol-induced peroxisome in Candida boidinii. (1) The structure, biosynthesis, and assembly pathway of 3 peroxisomal membrane proteins will be studied. Work will be completed to analyze the pathway of newly synthesized PMP 20 (peroxisomal membrane protein of 20 kDa) to the peroxisome and to sequence the gene encoding this protein. These studies will be extended to two other peroxisomal membrane proteins, probably PMP 31 and PMP 47. Monoclonal antibodies that are useful for immunoprecipitation, now available for PMP 20, will be raised against the other membrane proteins for this purpose. The genes encoding PMP 31 and 47 will be isolated from an existing genomic library and their sequences will be compared to try to identify common domains which may be important for the targetting of these proteins to the peroxisomes. (2) A system for expression in vivo of the isolated peroxisomal genes from Candida will be developed, in order to begin to study domains which are necessary for sorting. Expression and sorting of these genes in Saccharomyces cerevisiae will be investigated in order to take advantage of the knowledge and techniques of genetics in this organism. Alternatively, expression in Candida boidinii or another methylotroph will be explored. This may involve the development of auxotrophic mutants and vectors for transformation. This system will be the basis for studying the effects of gene alterations (such as deletions and fusions) on localization of the altered proteins.
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