The long-range goals of this project are to characterize important steps in regulation of synthesis, assembly, and function of the mitochondrial respiratory membrane through use of dormant and germinating spores of the fungi Botryodiplodia theobromae and Neurospora crassa. Objectives of this project include an analysis of the membrane system for translocation of peptides synthesized in the cytosol into the interior of the mitochondria, the coordinate activity of mitochondrial and nuclear genetic systems their separate synthesis of mRNAs for subunits of common enzymes, and a modification of certain enzyme subunit peptides or associated peptides with a derivative of pantothenate. The experimental use of dormant and germinating fungal spores may be especially productive in analysis of these issues in mitochondrial biogenesis. (1) Cloned cDNAs will be used in an in vitro transcription- translation system to generate pure, radiolabeled precursor peptides for specific subunits of cytochrome oxidase and mitochondrial ATPase. These precursors will be used to dissect the reversible inhibition of the peptide translocation system in the mitochondrial membranes of dormant and germinating spores of B. theobromae and to examine the normal recognition and translocation steps in mitochondria of N. crassa. (2) To help determine if expression of nuclear and mitochondrial genes for mitochondrial proteins may be regulated both transcriptionally and post- transcriptionally, cloned DNA probes for paired genes for subunits of cytochrome oxidase or ATPase will be used to compare kinetics o( accumulation and processing of transcripts in the dormant and germinating spores of N. crassa and during cellular response to heat shock. (3) Experiments are designed to help determine (a) if subunits of cytochrome oxidase are modified directly by the covalent addition of a pantothenate derivative, (b) if the modified peptide instead is an associated, auxiliary protein perhaps involved transiently in enzyme assembly, and (c) if this pantothenate modification could be involved in an addition of a fatty acid moiety to an enzyme subunit. Provided the cytochrome oxidase subunit is directly modified with pantothenate, the precursor of this subunit generated from a cloned gene will be employed to help determine the nature and function of this unusual type of peptide modification. This research project should contribute useful new understanding of regulation of cell dormancy and activation that is applicable to clinically important organisms whose life cycles include a phase of developmental arrest.
