The central hypothesis of this proposal is that phosphatidylglycerol (PG) and cardiolipin (CL) play defined and specific roles in the function of the mitochondria. Mutants of the yeast Saccharomyces cerevisiae lacking CL (crdlA lacking CL synthase) or PG and CL (pgslA lacking PG-phosphate synthase) have been made. Both mutants grow well on fermentable carbon sources, but the former grows poorly and the latter does not grow on non-fermentable carbon sources. Mitochondria of cells lacking PG and CL are unable to carry out oxygen-dependent energy production due to a defect in translation of one nuclear encoded (CQX4) and three mtDNA encoded (COX1-3) subunits of cytochrome c oxidase (Complex IV). The mtDNA-encoded cytochrome b (COB) subunit of Complex III (cytochrome bcl) is also not translated. Viable strains of yeast lacking either CL or PG and CL will be used as """"""""biological reagents"""""""" to determine the molecular basis for the requirement of these anionic phospholipids in normal mitochondrial function and thereby uncover new and novel roles for lipids in cell function.
Specific Aim 1 will utilize chimeric reporter genes fused to the transcribed but untranslated domains 5' of the Cox4p start codon to identify the cis- and trans-acting elements responsible for inhibition of translation of COX4 mRNA in the cytoplasm. Deletion analysis, protein- RNA hybridization, and identification of genes that suppress the lack of translation by plasmid library complementation or mutagenesis will be used.
In Specific Aim 2 a similar approach will be used to identify cis- and trans-acting elements that repress translation of the mRNA of chimeric reporter genes expressed by mtDNA of PG/CL-lacking cells.
Specific Aim 3 will test the hypothesis that the energy transducing machines of the inner mitochondrial membrane have a specific requirement for CL. Complexes Ill and IV have specifically bound CL, and they form a supramolecular complex postulated to tunnel electrons to molecular oxygen. The organization, stability, and function of this larger complex will be studied as a function of CL levels and growth conditions. Complex III will be purified from crdlA cells and studied in reconstituted proteoliposomes to determine the role CL plays in its structure and function. Defining the role these lipids play in normal mitochondrial function will shed light on the molecular basis for cellular dysfunction in diseases where these lipids are reduced.
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