The long term objective of this research is to determine the mechanism of action of the Mac1 protein in the yeast Saccharomyces cerevisiae. Mac1p is a copper-regulated transfactor that is required for the expression of the genes that encode the protein components of the high affinity copper uptake system in yeast. Chief among these genes is CTR1, which encodes the high affinity copper permease. We have localized the several functions of Mac1p to specific domains within the protein and, based on this domain-mapping propose an overall mechanism for how Mac1p is cycled between transcriptionally active and inactive states in a copper-dependent fashion. Each of these functions involves a specific intra- or intermolecular Mac1p interaction: with itself, with copper, with DNA, with components of the basal transcription machinery, and with a protein we have identified and produced, Mdr1p, that we propose is the lynch-pin of this cycling pathway. To test this hypothesis we propose three Specific Aims: I) Functional Analysis of the Domains of the Mac1 Protein, in which we will map the specific contacts essential for the protein-protein and protein-DNA interactions that determine Mac1p function. II) The Mdr1 Protein and Its Role in Copper Homeostasis, in which we will test possible mechanisms for how Mdr1p modulates the cycling of Mac1p between transcriptionally active and inactive states, in particular, in the termination of Mac1p activity by newly arrived copper. III) Copper Sorting and Sensing, in which we will test possible mechanisms for how newly arrived copper is sorted to Mac1p so as to terminate its activity at the CTR1 promoter. Saccharomyces cerevisiae is a remarkably versatile organism in which to systematically study the homeostatic mechanisms that eukaryotes have evolved to deal with environmental stress. As a free-living organism, yeast must be especially adept at altering the expression of uptake activities for both copper and iron so that there is sufficient cellular metal in times of deficiency while ensuring that excess metal is kept out of the cell in times of plenty. Mac1p is a novel, copper-dependent transfactor which must also be part of a signal transduction pathway that at some point must have a copper sensor (which could be Mac1p itself) to which copper must be sorted. In addition, Mac1p appears to be central to the physiologic interaction between copper and iron in yeast that parallels the copper-for-iron connection in humans. Thus, elucidation of the function and modulation of Mac1p in yeast will be broadly relevant to eukaryotic copper and iron metabolism in general.
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