The goals of this project are to understand how mitochondrial (mt) genes are coordinately expressed, leading to synthesis of mt membrane proteins, how the proteins are modified as they are assembled into functional complexes within the membranes, and how these membrane complexes are conserved functionally during cell exposure to physical stress, such as high temperature. Objectives of this project include an exploration of a type of mt gene regulation that occurs at the level of translation, a study of the mechanism and function of a newly discovered myristoylation of subunit 1 of cytochrome c oxidase (CCO), and an examination of the composition of the major import complexes of the outer mt membrane and how certain heat shock proteins, such as the alpha-crystallin-related hsp30, interact with and stabilize these complexes during stress. The organism used in this study, Neurospora crassa, is a genetically and biochemically well-characterized fungus, and its synchronized germinating conidiospores are especially suitable to study of mt biogenesis. (1)Mt proteins will be sought that bind to potential regulatory regions of mRNAs for subunits of CCO, and these proteins will be isolated and their interaction with the RNAs as well as with other proteins will be characterized. The cya-5 mutant of N. crassa, which contains a potentially translatable mt mRNA for CCO subunit 1 but does not accumulate the protein, is an attractive candidate for specific study of a defective mt translation factor. (2)The subunit 1 peptide of CCO contains an unusual myristoylation whose function is unknown. In vivo and in vitro experiments will be performed with both N. crassa and homologous bacterial peptides to determine the amino acid sequences necessary for this acylation as well as to characterize the mt enzymic system for myristoylation of this subunit. Antibodies to the subunit peptide and physical and biochemical studies will be used to help determine if this acylation is required for assembly or for function of the CCO complex. (3)Some heat shock proteins (hsp) localize to the outer mt membrane, and their function there is unknown. Preliminary evidence indicates that during exposure to the stress of heat shock these hsp associate with several large multi-component complexes of this membrane. Experiments will be performed to identify how hsp such as hsp30, hsp38, and hsp83 interact with and stabilize previously-identified components of the outer membrane import receptor complexes. Mutants of N. crassa deficient in hsp30 will be used to determine how the absence of this hsp affects mt membrane structures under stress. It is likely that the insight gained in this project from study of N. crassa will be applicable to the molecular biology of clinically important fungi, including those that cause mycoses in immunocompromised patients.
