INDUCTION OF AN ORGANELLE: THE YEAST PEROXISOME

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The assembly of the induced peroxisome in Candida boidinii yeast will be studied. This organelle is induced from an insignificant fraction of the cell to 20-80% of cytoplasmic volume (depending on strain) within 10 hours after incubation of cells in methanol. The induction phenomenon offers am excellent opportunity to study basic mechanisms of organellar assembly as well as to address the controversial issue of the origin of peroxisomes. The strategy will be to study the induction and assembly of a major peroximal matrix and membrane protein. The pathway of asssembly of peroxisomal proteins will be determined in vivo by a pulse-chase-fractionation-immunoprecipitation protocol. Antibody against alcohol oxidase, a very abundant matrix protein, is available, and antibody against peroxisomal membrane proteins (PMPs) will be generated. Preliminary studies indicate that this approach is feasible. The ssequence of induction of membrane and matrix proteins and their mRNAs will be determined. Knowledge of this order of events will be particularly useful at a later time when the function of the relevant PMPs are known, as well as for the elucidation of the mechanism of induction, a long range goal. A sequestration reaction in vitro of a peroxisomal protein by membranes will be developed to begin to dissect peroxisomal assembly at a molecular level and to answer questions of sorting signals and catalysis of assembly. It is hoped that this approach will eventually lead to the identification and study of the essential elements which are involved in these processes. These biochemical approaches will exploit the induction phenomenon and will begin to elucidate the coordination of events in the assembly of a newly synthesized organelle.