RUI: Effect of Amino Acid Substitutions on the Production, Localization, Processing, Stability and Function of Subunit II of Cytochrome c Oxidase in Yeast

Ithaca College, Ithaca NY

Investigators

Abstract

Cytochrome c oxidase (CcO) is an enzyme complex required for respiration in all eukaryotes and some prokaryotes. The enzyme complex is the terminal member of the electron transport chain and is responsible for transferring electrons from cytochrome c to molecular oxygen. The number of subunits in CcO is variable, ranging from three or four in bacteria to as many as 13 in the bovine enzyme complex. The three largest subunits, Cox1p, Cox2p, and Cox3p are encoded on mitochondrial DNA in eukaryotes and are closely related to the three subunits found in prokaryotes. Therefore, it is likely that these mitochondrially encoded subunits represent the catalytically significant subunits of the eukaryotic enzyme. Cox2p has been shown to carry a binuclear copper center, which is the initial site of electron entry from cytochrome c. To better understand the function of Cox2p, yeast strains with defects in cellular respiration due to mutations in the COX2 gene have been identified or created. For the site-directed mutations which have been created in this gene, the mutant strains will be characterized both genotypically and phenotypically. For each mutant strain, a large collection of revertant strains which have recovered respiratory function will be identified and characterized, and the nature of the genetic change which restores function will be determined. The mutant strains which bear amino acid substitutions will also be analyzed to determine the effect of each of the mutations on the localization, stability, processing, and assembly of Cox2p. Some of the mutations may interfere with one of these aspects but not others. For example, some mutations may produce a stable protein, which is assembled with the other Cox polypeptides into a complex, which is however, inactive. Other mutations may affect stability or localization to the correct cellular compartment. Because the Cox2p amino acid sequence is so highly conserved, information deduced about the function of this protein in yeast should help to elucidate the function of the protein in higher organisms. Such an understanding is of importance because of the profound effect of the protein on the basic energy metabolism of a broad range of organisms, from bacteria to primates. In addition to expanding our knowledge of this important protein, the project will involve undergraduate collaborators at all stages, including those of carrying out the actual experiments, drafting manuscripts, and presenting their results at scientific conferences. The hands-on training provided to students in the course of the research will help prepare them for graduate and professional school and will provide them with both technical and critical thinking skills.