The molecular events which control and coordinate the expression and assembly of a membrane bound enzyme complex normal conditions and the manner in which these events are perturbed in various membrane pathologies is at present poorly understood. We propose to study the role of nuclear genes in the assembly and function of the yeast mitochondrial ATPase complex, and membrane bound complex whose properties and composition have been well documented in recent studies. We shall isolate the structural genes for the ATPase complex subunits using either complementation of yeast nuclear ATPase mutants via transformation with a cloned yeast wild type DNA pool or using immunological detection of yeast ATPase antigenic determinants which are expressed in E. coli transformed with yeast DNA. The selected structural genes which are contained on yeast replicating plasmids will be prepared and analyzed by restriction endonuclease analysis and DNA sequencing methods to determine the structure of the ATPase subunit genes. These studies will not only provide a protein sequence from DNA sequence analysis for the subunits, but also make available cloned structural genes which will be used to specifically alter the coding sequences for different components of the ATPase complex. We shall compare derived protein sequences of the ATPase subunits in order to access the role of the specific sequences in the post translational segregation and processing events of mitochondrial membrane assembly. We propose to modify the isolated ATPase structural genes in vitro and introduce site specific mutations for various subunits of the enzyme by transformation of wild type yeast with integrating yeast plasmids containing altered DNA sequences. IN related studies we propose to investigate the mitochondrial membrane components which participate in the recognition and processing of ATPase precursor subunits during assembly. We plan to purify these components and examine the role of the components in the assembly of the ATPase complex using biochemical, genetic, immunological and recombinant DNA methodologies. The long term objectives of these combined studies is to further develop the yeast mitochondrial ATPase complex such that we will be able to alter the protein sequence of any subunit of the enzyme or the membrane components which participate in its assembly to ask specific questions about the assembly and/or function of these molecules in energy conserving or membrane assembly events.
