A. Gangar et al., Isolation and localization of a cytosolic 10 S triacylglycerol biosynthetic multienzyme complex from oleaginous yeast, J BIOL CHEM, 276(13), 2001, pp. 10290-10298
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.