Assembly of membranes in heart and yeast mitochondria

Mitochondria play a central role in oxidative metabolism by providing most of the ATP needed for the cell's survival. Maintenance of this vital function is critically dependent on a modest number of genes residing in the mitochondrial genome and the much larger pool of genetic information provided by the nucleus. We have studied a collection of nuclear respiratory defective mutants of Saccharomyces cerevisiae consisting of some 200 complementation groups that define much of the metabolism specific to mitochondria and the process by which this organelle increases in mass during cell growth and division. Approximately 75% of the genes defined by these mutants have been functionally characterized and we hope to have this aspect completed in the coming grant period. Within the purview of this proteomic project, however, we have also continued studies on the mechanism of assembly of complex membrane enzymes such as the H+-translocating ATPase, the bc1 complex and more recently mitochondrial ribosomes. During the last period substantial progress was made on several fronts including the pathway of ATPase biogenesis. In the coming period we intend to continue three areas of studies. First we will extend our functional analyses of Atp25p and Atp23p with the aim of better understanding the manner in which these chaperones promote, respectively, formation of the ATPase subunit 9 ring and interaction of subunit 6 with the ring. We will modify the mitochondrial genes for subunits 6, 8, and 9 so that they will be expressed with tags suitable for detection and affinity purification. This has already been done for subunit 6. These constructs will be used to characterize the early intermediates formed during assembly of the F0 sector. We will also continue to screen for additional ATPase genes of which we believe there remain more to be found. In related studies we will examine the roles of Cbp3p in assembly of the bc1 complex and of a new GTPase in maturation of the large subunit of mitochondrial ribosomes. The third area will exploit our recent finding that mitochondrial copper homeostasis is regulated by an Ssn6-Tup1 dependent signaling pathway. Two immediate questions of interest are 1) is this pathway involved in copper import into or distribution within mitochondria, and 2) what are the other signaling molecules in the pathway? Most of the yeast genes we have studied in the past have homologues in higher eukaryotic genomes and an increasing number have been shown to be involved in human neuromyopathies. Further characterizations of the mutants in our collection will enlarge on this information and thereby improve the yeast model as it applies to human mitochondrial diseases.