Regulation of cardiolipin biosynthesis in yeast.

Cardiolipin (CL) is a phospholipid which is found only in mitochondrial membranes in eucaryotic cells. CL biosynthesis was studied in Saccharomyces cerevisiae and Schizosaccharomyces pombe to further understand the regulation and function of this phospholipid. The second enzyme of the CL pathway, phosphatidylglycerolphosphate (PGP) phosphatase, was characterized in the yeast S. cerevisiae biochemically and with respect to regulation by inositol and respiratory versus fermentative growth. Because experiments showed PGP phosphatase was not regulated, further studies focused on the first enzyme of the CL pathway, PGP synthase (PGPS), which is highly regulated in S. cerevisiae. To identify the yeast PGPS gene, a genetic screen was developed to isolate S. cerevisiae mutants unable to survive without a plasmid that expresses the E. coli pgsA gene, which encodes PGPS activity. The bacterial gene was expressed in yeast, as measured by enzyme activity. However, no yeast PGPS mutants were isolated using this method. A second approach to isolate the PGPS gene, purification of the PGPS protein, was initiated. PGPS from S. cerevisiae was inactivated by detergents, precluding biochemical studies of this protein. Therefore, biochemical and regulatory analysis of PGPS in S. pombe was started. As in S. cerevisiae, PGPS activity in S. pombe is regulated by inositol and this regulation requires a functional phophatidylcholine pathway. Preliminary studies showed that PGPS in S. pombe could be solubilized by the detergent Triton X-100. PGPS was purified 10,377-fold over cell extracts using a combination of cation exchange, hydroxylapatite, and gel filtration chromatography. The highly purified enzyme preparation was maximally active at pH 7.5 in the presence of Triton X-100. The curve for dependence of activity on the phospholipid substrate, CDP-DG, was sigmoidal, suggesting this phospholipid acts as a substrate and activator of the enzyme. The most effective lipid activator was CL, in the presence of which the curve for the dependence of activity on CDP-DG was hyperbolic. Both phosphatidylinositol (PI) and inositol inhibited activity of purified PGPS.Ph.D.Biological SciencesCellular biologyMicrobiologyMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/129069/2/9319559.pd