The bioenergetic 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 the subunit composition ?3?3???. 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 ? and ? subunits of the F1 accumulate as aggregated proteins inside the organelle. In the next funding period we will continue our efforts to provide detailed information about the chaperone actions of Atp11p and Atp12p. Studies under Aim 1 focus on the interactions of these proteins with natural and model ligands. Structure/function relationships in the chaperone domain of Atp11p will be probed through the use of a fluorescent compound that has been shown to bind in this region, and through site-directed mutagenesis of conserved sequence elements; the ramifications of the latter will be assessed both in vivo and in vitro. In other work, Atp11p, Atp12p, and their substrate proteins (the F1 ? and ? subunits) will be examined for actions as mediators of protein-protein interactions. Studies will also be pursued to determine if there are additional proteins in mitochondria that act in concert with Atp11p and Atp12p. The goal of Aim 2 is to solve the three-dimensional structure of Atp11p and of Atp12p by means of X-ray crystallography. The F1 chaperones are proposed to interact with their protein substrates via complementary surfaces. Under this point of view, the structural analysis is expected to play a major role in enabling us to solve the mechanism of Atp11p and Atp12p.
Aim 3 focuses on the process of F1 subunit aggregation that occurs under conditions in which Atp11p or Atp12p is not present. As it is now known that Atp11p and Atp12p are chaperone proteins of human cells, detailed information about what occurs when these proteins are deficient or defective is of interest.
