Significant reduction of mitochondrial cardiolipin (CL) has been postulated to compromise directly cytochrome oxidase function, the ADP/ATP translocator, phosphate translocator, ATP synthase, palmitoyl carnitine transferase, and carnitine translocase. Inhibition of any of these processes would lead to mitochondrial dysfunction particularly in the process of oxidative phosphorylation. Compromising terminal oxidation has been shown to accentuate both short term and long term oxidative damage to tissue because of the build up of highly reactive intermediates generated by the electron transport chain leading to even further damage. With aging the cholesterol to phospholipid ratio of heart muscle mitochondrial increases due to loss of CL which has been associated with increases in membrane rigidity. To date there is no genetic evidence for any of the critical roles with which CL has been associated, and the lack of mutants in the synthesis of CL in eukaryotic cells has made it difficult to reconcile in vitro observations of the role of CL with in vivo processes. We have isolated the genes (PGSI and CLS1) from Saccharomyces cerevisiae that encode the phosphatidylglycerol-(PG) phosphate (P) and CL synthases, which are responsible for the synthesis of CL in mitochondria. By making mutants null in the these genes, the requirement for PG/CL for both cell viability and mitochondrial function will be established. Using these null mutants and strains in which the expression of these genes are artificially regulated, the cellular processes dependent on PG and/or CL will be identified and their requirement for these lipids will be detailed at the molecular level. Since CL appears to be a critical for phospholipid to mitochondrial function, understanding the regulation of its synthesis is important to a better understanding of the relationship between variation in CL levels and cellular dysfunction. The regulation at the level of transcription and translation of these genes will be investigated to gain insight into CL homeostasis. Additional insight into CL regulation will be obtained by purifying and characterizing the enzymological properties of the PG-P synthase, which is the rate limiting enzyme in CL synthesis, as well as the CL synthase, the terminal step in CL synthesis. Given the complexity of molecular genetic and biochemical studies in somatic cells and the similarity of apparent CL function in all eukaryotic cells, the detailed studies of CL synthesis and function in yeast will lead to a clearer understanding of the function of this phospholipid in all eukaryotic cells.
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