PURIFICATION AND CHARACTERIZATION OF PHOSPHATIDYLGLYCEROLPHOSPHATE SYNTHASE FROM SCHIZOSACCHAROMYCES-POMBE

The enzyme CDP-diacylglycerol:sn-glycerol-3-phosphate 3-phosphatidyltr ansferase (phosphatidylglycerolphosphate synthase; PGPS(4); EC 2.7.8.5 ) is located in the mitochondrial inner membrane and catalyzes the com mitted step in the cardiolipin branch of phospholipid synthesis. Previ ous studies revealed that PGPS is the most highly regulated enzyme in cardiolipin biosynthesis in both Saccharomyces cerevisiae and Schizosa ccharomyces pombe, In this work, we report the purification to homogen eity of PGPS from S. pombe, The enzyme was solubilized from the mitoch ondrial membrane of S. pombe with Triton X-100. The solubilized enzyme , together with the associated detergent and intrinsic lipids, had a m olecular mass of 120 kDa, as determined by gel filtration. The enzyme was further purified using salt-induced phase separation, gel filtrati on, and ionic exchange, hydroxylapatite, and affinity chromatographies , The procedure yielded a homogeneous protein preparation, evidenced b y both SDS-polyacrylamide gel electrophoresis (PAGE) and agarose isoel ectric focusing under nondenaturing conditions. The purified enzyme ha d an apparent molecular mass of 60 kDa as determined by SDS-PAGE. The enzyme showed a strong dependence on lipid cofactors for activity in v itro. While both phosphatidic acid and CDP-diacylglycerol appeared to be activators, the most significant activation was observed with cardi olipin, The possible physiological significance of the lipid cofactor effect is discussed, This is the first purification of a eucaryotic PG PS enzyme to date, and the first purification of a phospholipid biosyn thetic enzyme from S. pombe.