Robert Poyton - Professor

Our studies address (1) gene expression, (2) targeting and  assembly of membrane proteins, (3) cellular energy production,  and (4) oxidative stress. We use baker’s yeast, Saccharomyces cerevisiae, and employ genetics, genetic engineering with cloned genes, protein biochemistry, and in vitro gene expression systems. Our model for these studies is cytochrome c oxidase, a multi-meric protein of the inner mitochondrial membrane that is composed of three subunit polypeptides encoded by mitochondrial  genes (COX1-3) and six subunits encoded by nuclear genes  (COX4-9).

Gene Expression. Two aspects of gene expression are under study. First, we are examining how O2 activates transcription of COX5a and COX6 and represses transcription of COX5b. Our current goals are to identify the intracellular O2 sensor that translates O2 concentration into an effect on transcription and understand how the “O2 switch” works, locate O2 responsive promoter elements, identify proteins that bind to these O2 responsive elements, and determine how the O2 sensing pathway affects transcription. Second, we are studying how gene expression in the nucleus is coordinated with gene expression in the mitochondrion. Current studies are focused on how the mitochondrial genome regulates the expression of the nuclear COX genes. Recently, we have discovered that a mitochondrial gene is required for phosphorylation  of a transcription factor that regulates the nuclear COX  genes. We are now attempting to elucidate the signal transduction  pathway from the mitochondrion to the nucleus.

Protein Assembly. The formation of cytochrome oxidase requires the import of nuclear-coded subunits from the cytosol, the export of mitochondrially coded subunits from the matrix, and an assembly pathway that brings both sets of subunits together. We are using newly developed in vivo and in vitro systems and “assembly defective” mutants to examine the sequence in which subunits come together during assembly and identify a new type of molecular chaperone that is involved in cytochrome c oxidase assembly.

Energy. Recently we have developed methods for studying the electron transfer reactions of cytochrome oxidase in whole cells.  Now we plan to use site-directed mutagenesis to study the structural features of its nuclear-coded subunits that are important for function in vivo. In addition, we have developed a  heterologous complementation system with yeast for assaying  human cytochrome c oxidase subunit function and have used it  to clone human cytochrome c oxidase subunits and cytochrome  c oxidase specific molecular chaperones. Eventually, we hope to  use this system for assaying subunit dysfunction in a growing  number of human diseases (myopathies and neuropathies) that  have been linked to defects in cytochrome c oxidase.