The synthesis and assembly of specific components of the mitochondrial respiratory membrane will be studied during spore formation and spore germination of the fungi Neurospora crassa and Botryodiplodia theobromae. The dormant spores of Botryodiplodia (in contrast to those of Neurospora) contain mitochondria that are deficient in certain respiratory enzyme complexes. The activities of these enzymes, however, are required for spore germination, and they are reassembled rapidly by mobilization of preserved subunits from the cytoplasm (cytochrome c oxidase) or by de novo synthesis of subunits from a preserved mRNA (ATPase). One objective of this proposed research project is to study the mechanism of translocation of cytoplasmic subunits of cytochrome c oxidase (obtained from a cell-free mRNA translation system or from the cytoplasm of spores) into mitochondria isolated from dormant and germinated spores. Experiments will be performed to determine if the exclusion of these oxidase subunits from mitochondria of dormant spores (and their accumulation in the cytoplasm) could be caused by a reversible inhibition or block of the translocation system as a programmed step of developmental arrest. A second objective of this research project is to study the transcription of nuclear genes for several subunit proteins of ATPase and cytochrome c oxidase. The synthesis of the mRNA for these proteins will be measured (by hybridization to a cloned yeast gene or cloned cDNA) during spore formation and germination to learn how transcription may be affected by stage-specific metabolic events and restrictive germination conditions. These hybridization probes also will be used to determine how and in what cellular fraction the preserved mRNAs for the enzyme subunits are stored in the cytoplasm of the dormant cells. This study of mitochondrial biogenesis in spores should yield new insight into the mechanism of respiratory membrane assembly that otherwise is not readily available in other types of experimental organisms where steps of gene transcription, mRNA translation, and enzyme assembly are temporally much less distinct processes. This study also should contribute useful new understanding of the regulation of cell dormancy and activation that will be applicable to clinically important organisms (bacterial and fungal spores, insect eggs and larvae protozoan and parasitic worm cysts) which include in their life cycles a phase of dormant or developmental arrest.
