Abstract
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.
Highlights
Growth and TAG Synthesis in R. glutinis—The growth of the oleaginous yeast cells was monitored by both A600 and colonyforming units at 30 °C (Fig. 1, A and B)
To determine the rate of TAG biosynthesis in R. glutinis, cells were metabolically labeled with [14C]acetate, and its incorporation into free fatty acids, DAG, TAG, and PL was measured at various growth intervals (Fig. 1E)
The analysis revealed that the lysophosphatidic acid (LPA) acyltransferase, phosphatidic acid (PA) phosphatase, and DAG acyltransferase activities were associated with one fraction
Summary
The hexane layer was removed and concentrated, and the lipids were separated on silica gel G thin layer plates developed with petroleum ether/diethyl ether/acetic acid (70:30:1, v/v/v). The amount of labeled acyl-ACP formed was determined by spotting the reaction mixture on Whatman No 3MM and washing the filter paper with chloroform/methanol/acetic acid (3:6:1, v/v/v) followed by liquid scintillation counting. Sucrose Density Gradient—The soluble fraction (75 mg) or the purified complex (25 g) was layered onto a 10 –30% linear sucrose gradient containing 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, and 0.1 M NaCl. The tubes were centrifuged for 18 h at 200,000 ϫ g (Beckman SW 41 rotor), and fractions (1 ml) were collected and assayed for enzyme activities. E, metabolic labeling of yeast cells with [14C]acetate and its incorporation into TAG, DAG, free fatty acids, and PL at various time intervals was performed. Incorporation and analysis of labeled lipids was carried out as described under “Experimental Procedures.”
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