Isolation and localization of a cytosolic 10 S triacylglycerol biosynthetic multienzyme complex from oleaginous yeast

Triacylglycerol is one of the major storage forms of metabolic energy in eu karyotic cells. Biosynthesis of triacylglycerol is known to occur in membra nes. We report here the isolation, purification, and characterization of a catalytically active cytosolic 10 S multienzyme complex for triacylglycerol biosynthesis from Rhodotorula glutinis during exponential growth. The comp lex was characterized and was found to contain lysophosphatidic acid acyltr ansferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, a cyl-acyl carrier protein synthetase, and acyl carrier protein. The 10 S tri acylglycerol biosynthetic complex rapidly incorporates free fatty acids as well as fatty acyl-coenzyme A into triacylglycerol and its biosynthetic int ermediates, Lysophosphatidic acid acyltransferase, phosphatidic acid phosph atase, and diacylglycerol acyltransferase from the complex were microsequen ced. Antibodies were raised against the synthetic peptides corresponding to lysophosphatidic acid acyltransferase and phosphatidic acid phosphatase se quences. Immunoprecipitation and immunolocalization studies show the presen ce of a cytosolic multienzyme complex for triacylglycerol biosynthesis, Che mical cross-linking studies revealed that the 10 S multienzyme complex was held together by protein-protein interactions. These results demonstrate th at the cytosol is one of the sites for triacylglycerol biosynthesis in olea ginous yeast.