The proper absorption, distribution and utilization of metals in cells is an important yet poorly understood process. The requirement for metals in a large number of biochemical processes and the toxicity of high intracellular metal concentrations necessitates the existence of a finely balanced intracellular metal homeostatic process. This application outlines plans to investigate in detail one aspect of intracellular metal homeostasis in the baker's yeast Saccharomyces cerevisiae. The role the yeast ACE1 gene plays in sensing high intracellular copper concentrations and transmitting this information to activate the expression of a copper-detoxification gene will be investigated. The site of action of the ACE1 gene product will be determined by intracellular localization studies using indirect immunofluorescence and by following the fate of an ACE1-beta-galactosidase fusion protein expressed in yeast cells. Regions of the ACE1 gene product important for function will be determined by creating random mutations in the wild-type ACE1 gene and returning the altered derivatives to yeast cells to test function in vivo. A possible direct interaction between the ACE1 gene product and yeast metallothionein transcriptional control sequences will be tested by synthesizing the ACE1 protein in heterologous systems and conducting DNA binding studies. These studies should provide a sound basis for determining how yeast cells maintain appropriate intracellular copper concentrations and should contribute to a basic understanding of cellular metal homeostatic mechanisms.
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