Mitochondrial Copper Transport in Saccharomyces cerevisiae
Type of Degreedissertation
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Saccharomyces cerevisiae contains a copper pool within the mitochondrial matrix that is required for assembly and activity of cytochrome c oxidase. Matrix copper exists bound to an anionic, fluorescent molecule known as the copper ligand (CuL). The CuL is imported into isolated mitochondria in a time dependent, saturable manner. Using silver as a copper competitor, we screened mitochondrial carrier family mutants and identified PIC2 as a candidate transporter. The pic2∆ mutants grew poorly on copper-deficient, non-fermentable medium supplemented with silver, a phenotype that was exacerbated by expression of a matrix targeted competitor protein. Copper and copper uptake was deficient in mitochondria from pic2∆, and Pic2 expressed in Lactococcus lactis mediated uptake of copper given both as the CuL and as copper salts. Therefore, we propose that Pic2 mediates copper import into the mitochondrial matrix. Here, it is shown that deletion of PIC2 caused a defect in expansion of the mitochondrial CuL pool and uptake of the CuL complex as measured by thin layer chromatography (TLC). Additionally, a physical interaction between Pic2 and the CuL was demonstrated using fluorescence anisotropy. Taken together, these data suggest that Pic2 mediates import of the entire CuL complex. While deletion of PIC2 caused a decrease in mitochondrial copper and CuL, it was not completely depleted. We looked to another mitochondrial carrier family gene, MRS3, previously identified as being involved in high affinity iron uptake, as potential member of the copper import pathway. While deletion of this gene led to copper related growth phenotypes and a decrease in mitochondrial copper expansion, it did not affect mitochondrial copper uptake. Mrs3 expressed in a heterologous system, though capable of importing iron, did not mediate copper import. Therefore, MRS3 is a modifier of PIC2 and has some indirect effect on mitochondrial copper import.