Eukaryotic cells satisfy their energy needs mainly with the ATP produced by metabolic pathways housed in mitochondria. Propagation of this organelle, once a free living organism, depends largely on genes, which in the course of evolution have been transferred to chromosomal DNA. In this proposal, a collection of respiratory defective nuclear mutants of Saccharomyces cerevisiae will be enlisted to examine questions related to the mechanisms that govern and regulate the maintenance of respiratory competent mitochondria. Three areas of studies will be pursued. The first will probe how F1-F0 ATPase, a universal device for ATP synthesis, is assembled. The functions of two genes, ATP10 and ATP22, required for assembly of F0, will be studied by combined biochemical and genetic approaches. Concurrently, the mutant collection will be screened for additional ATPase-assembly genes. These studies will enlarge the number of presently known ATPase-specific assembly factors and clarify still poorly understood aspects of ATPase biogenesis. The second objective is to clarify the roles of a new class of genes required for expression of a functional mitochondrial protein synthesis machinery. MTG1 is an example of a gene recently shown to function in ribosome assembly. Future studies on this group of genes will make use of ts mutants to ascertain if they are affected at the level of the machinery or the translation process itself. The state of ribosome assembly will be determined and the gene targets examined by suppressor analysis and other means. The third goal is to enlarge on our still fragmentary information about the pathways that modulate the oxidative capacity of mitochondria. As a starting point, an ssn6 mutant selectively impaired in respiration will be exploited to elucidate the role of the Ssn6p/Tup1p co-repressor complex and cAMP in regulating expression of mitochondrial metabolism. As in prior years, the possible involvement in human pathologies of genes identified from studies of the yeast model, will be assessed in collaborations with other laboratories engaged in screening patients with non-maternally inherited mitochondrial diseases.
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