COPPER METALLO BIOCHEMISTRY IN SACCHAROMYCES CEREVISIAE

酿酒酵母中的铜金属生物化学

• 批准号: 2701550
• 负责人: DANIEL J. KOSMAN
• 金额: $ 20.3万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2701550
• 关键词: Saccharomyces cerevisiae; X ray crystallography; biological models; biological signal transduction; copper; enzyme activity; fungal genetics; gene complementation; gene expression; membrane transport proteins; metal metabolism; metalloproteins; molecular cloning; oxidative stress; oxidoreductase; protein structure function; recombinant proteins; site directed mutagenesis; superoxide dismutase

The long-term objective of this research is to achieve a detailed understanding of the mechanisms which a eukaryote evolved to accumulate the essential trace nutrient copper, to regulate this accumulation, and to make this redox active metal available for metallating apo-cuproproteins. We intend to continue to exploit the characteristics of the budding yeast, Saccharomyces cerevisiae, which make it unique as a model system to establish possible mechanisms of Cu-handling and its regulation. We have demonstrated biochemically that in the initial step of Cu-accumulation, Cu(II) is reduced by at least two separate reductase activities in the yeast plasma membrane. The expression (synthesis) of these reductases, which support both Cu(II) and Fe(III) accumulation by yeast, requires a protein, Mac1p, whose gene we have cloned. Mac1p also is required for the expression of genes associated with carbon catabolite control, peroxidative stress, and heat shock. Based on its primary sequence, Mac1p may be a metalloprotein. In Specific Aims I and II we will test two hypotheses about Mac1p: 1) that it serves as a primary sensor in a signalling pathway which results in specific gene expression and 2) that a metal ion - either copper or iron - in Mac1p serves as sensor and switch in this signal transduction. Wild type Mac1p will be overexpressed and characterized to determine whether it contains a metal ion, and the spectroscopic and electrochemical properties of the metal-binding site will be evaluated. Whether Mac1p modulates gene expression by binding to (an)other protein and/or to DNA will be determined by in vivo and in vitro approaches. The in vivo function of site-directed Mac1p mutants will be assessed; loss-of-function mutants will be characterized in vitro. We have demonstrated that one of the two reductase activities in the membrane is associated with the product of the FRE1 gene and that this enzyme can use both Fe(III) and Cu(II) as substrate. In Specific Aim III we will test by genetic and biochemical means the hypothesis that the other reductase is a unique Cu(II)-specific enzyme. A mutant in this locus will be generated and the wild type gene cloned by complementation. We have demonstrated that correct intracellular Cu- (and Fe-) trafficking requires the acidification of the yeast vacuole(s). In Specific Aim IV we will test the dual hypothesis that this organelle is the initial site of intracellular Cu-accumulation, and that delivery of copper to apo- cuproproteins occurs from vacuolar stores. The proposed research is based on testable models of copper metabolism and metal-dependent gene regulation in S. cerevisiae. It makes full use of the fact that this organism remains unique among eukaryotes as a cell system in which all of the tools of classical and molecular genetics, cell biology and biochemistry can be used systematically. The details of how S. cerevisiae metabolizes copper may be in part specific to this eukaryote. However, our view is that the aqueous chemistry of copper dictates how this toxic nutrient is dealt with by an aerobic cell, and that the mechanisms suggested by our proposed studies in yeast will serve as paradigms for the design of productive studies in other cell types.

本研究的长期目标是实现详细的