Eukaryotic cells contain as many as a dozen distinct subcellular compartments. Each of these compartments or organelles carries out a specialized set of functions that are crucial to cell viability. Indeed, many inherited genetic disorders in man are known to be caused by alterations in normal organelle functioning (e.g., the lysosomal storage diseases). The molecular mechanisms responsible for the accurate delivery of proteins to their correct organelle destinations remain largely unknown. Our research goal is to understand how certain mitochondrial and lysosomal (vacuolar) proteins in yeast are selectively and efficiently transported from their site of synthesis in the cell cytoplasm to their unique site of function in either the mitochondrion or the vacuole. The technique of gene fusion is being used to map the sequence or conformational signals in the nuclear encoded mitochondrial F1- ATPase beta-subunit protein and two vacuolar proteins, carboxypeptidase Y and Pep4, that allow these proteins to be properly sorted to their respective organelle destinations. Gene fusions have been constructed between the yeast genes that encode these proteins and either the E. coli lacZ gene or the yeast SUC2 gene (codes for the secreted enzyme invertase). Sequences identified in these mitochondrial and vacuolar proteins that are capable of redirecting either E. coli beta-galactosidase or yeast invertase to the mitochondrion or vacuole will be modified by site-directed mutagenesis. The effect these mutant sequences have on proper delivery of both the fusion proteins and the prototypic proteins will be analyzed. These studies should help to define the information content of a few representative protein sorting signals. Yeast mutants altered in their ability to properly recognize these protein sorting signals have been selected by exploiting certain phenotypes conferred to yeast by the gene fusion constructs. The genes affected in these mutants may code for components of the cellular machinery that function to interpret the protein sorting signals and direct these proteins to their proper organelle destinations. These genes will be cloned by complementation in yeast. Antisera will be raised against the products of these genes after overexpression in bacteria. These antisera will then be employed to analyze the site of cellular residence of these proteins in yeast (using immunofluorescence and cell fractionation techniques) and their mechanism of biogenesis.
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