A fundamental problem of cell biology and biochemistry is elucidation of the mechanisms of membrane biogenesis. While considerable information has accumulated to describe the mechanisms of intracellular membrane protein transport, little detailed biochemical information is available to describe lipid transport processes. These lipid transport processes are essential for cell growth, development, replication and homeostasis. The long term goals of this project are to define intracellular lipid transport at the molecular level. This proposal will address the problem of lipid transport in membrane assembly using both genetic and biochemical methods. The primary experimental system to be used in these studies is the yeast Saccharomyces cerevisiae. In this organism it is now possible to select for mutants defective in the interorganelle transport of phosphatidylserine and phosphatidylethanolamine. Using these newly available methods for mutant isolation we will obtain detailed molecular information about organelle biogenesis by I) cloning and sequencing the genes regulating the transport of phosphatidylserine to the locus of Phosphatidylserine decarboxylase 2; 2) isolating mutants defective in transporting phosphatidylserine to the locus of Phosphatidylserine decarboxylase 1, and then cloning and sequencing the genes that complement these mutants; 3) isolating mutants defective in the export of phosphatidylethanolamine out of the mitochondria, and then cloning and sequencing the genes that complement these mutants. The yeast mutants will also be used to isolate the corresponding human genes by complementation. To study the function of the isolated genes, they will be overexpressed using baculovirus vectors and the resultant gene products will be used to reconstitute lipid transport in permeabilized yeast and mammalian cells. These studies will provide new information about the genetics and molecular biology of membrane formation and organelle biogenesis.
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