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 eukaryotic cells. Biosynthesis of triacylglycerol is known to occur in membranes. 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 complex was characterized and was found to contain lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, acyl-acyl carrier protein synthetase, and acyl carrier protein. The 10 S triacylglycerol biosynthetic complex rapidly incorporates free fatty acids as well as fatty acyl-coenzyme A into triacylglycerol and its biosynthetic intermediates. Lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, and diacylglycerol acyltransferase from the complex were microsequenced. Antibodies were raised against the synthetic peptides corresponding to lysophosphatidic acid acyltransferase and phosphatidic acid phosphatase sequences. Immunoprecipitation and immunolocalization studies show the presence of a cytosolic multienzyme complex for triacylglycerol biosynthesis. Chemical cross-linking studies revealed that the 10 S multienzyme complex was held together by protein-protein interactions. These results demonstrate that the cytosol is one of the sites for triacylglycerol biosynthesis in oleaginous yeast.
Gangar
A
A
Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
Karande
A A
AA
Rajasekharan
R
R
D000217:Acyltransferases / Q000737:chemistry; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002455:Cell Division; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D051048:Diacylglycerol O-Acyltransferase; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000302:isolation & purification*; D010078:Oxazines / Q000494:pharmacology; D010711:Phosphatidate Phosphatase / Q000737:chemistry; D011233:Precipitin Tests; D012248:Rhodotorula / Q000201:enzymology*; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D014280:Triglycerides / Q000096:biosynthesis*
D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D009097:Multienzyme Complexes; D010078:Oxazines; D014280:Triglycerides; C008619:Nile Blue; D000217:Acyltransferases; D051048:Diacylglycerol O-Acyltransferase; C018764:2-acylglycerophosphate acyltransferase; D010711:Phosphatidate Phosphatase