Yeast peroxisomes are excellent model systems both for understanding general mechanisms of organelle biogenesis and for studying the basis of catastrophic perioxisomal diseases. We will take advantage of the abundance and simplicity of peroxisomes in Candida boidinii and the powerful genetics of Saccharomyces cerevisiae to elucidate three fundamental and interrelated functions of the peroxisomal membrane in protein import and organelle assembly. (1) We will determine the import pathway of an oligomeric peroxisomal matrix protein. Peroxisomes can import large oligomeric proteins and even inorganic particles. Alcohol oxidase (AO), a homo-octamer, will be used as a model protein to study import. The steps of AO import will be defined in detail using three approaches: coexpression of targeted and untargeted subunits in S. cerevisiae, pulse-chase analysis in C. boidinii, and microinjection in animal cells. We shall determine the intracellular site of octamerization and identify the members of an import complex described previously. We shall determine whether import of AO involves subunit dissociation and reassociation, and whether it occurs through a vesicular compartment. (2) We will identify the critical components for the assembly of an integral peroxisomal membrane protein (Pmp). We have described the first targeting signal on an integral Pmp, termed the """"""""loop"""""""" of Pmp47. The loop is different from signals on matrix proteins. We will select mutants that are unable to target or assemble a chimeric protein containing the loop as well as mutants that suppress altered loop sequences. We will determine the ability of the mutants to assemble an unrelated peroxisomal membrane protein, Pmp27, as well as to important matrix proteins. The genes corresponding to the mutations will be isolated by complementation. Interactions of their encoded proteins with each other and with known Pmps will be identified to elucidate their functions. (3) We will determine the mechanism by which Pmp27 causes peroxisomal shape changes and organellar proliferation. Disruption of PMP27 leads to the formation of one or two giant peroxisome per cell, and overexpression of the gene causes hyperproliferation of the organelle. We will confirm a function of Pmp27 in determining peroxisomal size, shape and number by additional expression experiments. We will test the hypothesis that Pmp27 interacts directly with the membrane to change its shape by studying the interaction of pure Pmp27 with membranes and synthetic bilayers. The regulation of Pmp27 function by homo-dimerization or interaction with Per8p, another membrane protein that may be required for peroxisomal proliferation, will be tested.
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