Transition Metal Homeostasis in Saccharyomyces cerevisiaeEPA Grant Number: U915646
Title: Transition Metal Homeostasis in Saccharyomyces cerevisiae
Investigators: Portnoy, Matthew E.
Institution: The Johns Hopkins University
EPA Project Officer: Lee, Sonja
Project Period: September 1, 1999 through August 1, 2002
Project Amount: $68,000
RFA: STAR Graduate Fellowships (1999) RFA Text | Recipients Lists
Research Category: Fellowship - Molecular Biology/Genetics , Academic Fellowships , Biology/Life Sciences
The objectives of this research project are to: (1) investigate how the budding yeast Saccharomyces cerevisiae uses the essential transitional metals, copper and iron, for cellular function; and (2) determine how the cell protects itself against these Fenton-reactive metals.
ATX1 protein delivers copper from an unknown source to a P-type Outpace (CCC2) in the Golgi for eventual incorporation into a multicopper oxidate involved in high-affinity iron uptake for the cell. Additionally, ATX1 serves as an antioxidant, protecting the cell against superoxide anion effects in cells lacking superoxide dismutase. Our approach is to perform a structure-function analysis using the three-dimensional, x-ray crystal structure of the ATX1 protein to select residues for site-directed mutagenesis. We will determine which amino acid residues are important for the copper delivery role of ATX1 and which are responsible for its antioxidant activity, using assays distinct for those functions. To determine the upstream source of copper for ATX1, a reverse-genetic approach will be used. Genes known to be involved in copper metabolism will be deleted in the genome, and the effect on ATX1 function will be assessed. Gene deletions that affect the copper status of ATX1 are candidates for the upstream copper source. SMF3 was identified as a homolog to the NRAMP family of mammalian metal transporters, by sequence analysis of the recently completed genome of S. cerevisiae. A classical molecular-genetic approach will be used to determine the potential metal regulation of SMF3 and the function of the gene product. Using Western blot analysis, indirect immunofluorescence, and atomic absorption spectroscopic analysis, the function and regulation of the SMF3 gene product will be determined.