The energy needs of aerobic cells are met principally through the action of the F1F0 ATP synthase, which catalyzes ATP synthesis during oxidative phosphorylation. The catalytic unit of the enzyme (F1) is a multimeric protein of subunit composition alpha3beta3gamma-delta-epsilon. Work in our laboratory, which employs the yeast Saccharomyces cerevisiae as a model system for studies of mitochondrial function, has provided evidence that assembly of the F1 oligomer in mitochondria requires two molecular chaperone proteins called Atp11p and Atp12p. Without these proteins, the alpha and beta subunits of the F1 accumulate as aggregated proteins inside the organelle. Previous studies have shown that Atp11p binds the F1 beta subunit and Atp12p binds the F1 alpha subunit. A subset of studies under Aim 1 (Characterization of intermediates in the F1 assembly pathway) are designed to determine what happens to the F1 subunits following their release from the chaperone proteins. Structure/function relationships in Atp11p and Atp12p will be probed by chemical modification and mutagenesis. Other studies under Aim 1 focus on the relationship of Atp11p and Atp12p with the Hsp60/Hsp10 folding machinery of mitochondria. In work under Aim 2 (Identification of protein protein interactions durinq F1 assembly) we will examine Atp11p, Atp12p, and the F1 alpha and Beta subunits for actions as mediators of protein-protein interactions during the process of F1 assembly. We will also characterize mutations in the genes for the alpha and beta subunits that cause an assembly defective phenotype and screen for additional proteins in mitochondria that may act in concert with Atp11p and Atp12p. Work under Aim 3 (Engineerinq of Atp11p and Atp12p for structural studies) is a collaboration between our group and two laboratories of structural biology to determine the three dimensional structure for Atp11p and for Atp12p by means of X-ray crystallography and nuclear magnetic resonance.