Synthesis of triacylglycerols by the acyl-coenzyme A:diacyl-glycerol acyltransferase Dga1p in lipid particles of the yeast Saccharomyces cerevisiae

Abstract

The terminal step of triacylglycerol (TAG) formation in the yeast Saccharomyces cerevisiae is catalyzed by the enzyme acyl-CoA:diacylglycerol acyltransferase (DAGAT). In this study we demonstrate that the gene product of YOR245c, Dga1p, catalyzes a major yeast DAGAT activity which is localized to lipid particles. Enzyme measurements employing a newly established assay containing radioactively labeled diacylglycerol (DAG) as a substrate and unlabeled palmitoyl-CoA as a cosubstrate revealed a 70- to 90-fold enrichment of DAGAT in lipid particles over the homogenate but also a 2- to 3-fold enrichment in endoplasmic reticulum fractions. In a dga1 deletion strain, the DAGAT activity in lipid particles is dramatically reduced, whereas the activity in microsomes is affected only to a minor extent. Thus, we propose the existence of DAGAT isoenzymes in the microsomal fraction. Furthermore, we unveiled an acyl-CoA-independent TAG synthase activity in lipid particles which is distinct from Dga1p and the phosphatidylcholine:DAGAT Lro1p. This acyl-CoA-independent TAG synthase utilizes DAG as an acceptor and free fatty acids as cosubstrates and occurs independently of the acyl-CoA synthases Faa1p to Faa4p. Based on lipid analysis of the respective deletion strains, Lro1p and Dga1p are the major contributors to total cellular TAG synthesis, whereas other TAG synthesizing systems appear to be of minor importance. In conclusion, at least three different pathways are involved in the formation of storage TAG in the yeast.

FIG. 1.

Dependence of DAGAT activity on a specific ionic environment. DAGAT assays without ions (open squares) and in the presence of K⁺ (open triangles), Mg²⁺ (filled squares), or both ions (filled triangles) were performed with microsomes or lipid particles of the wild-type strain X2180-1A as enzyme sources, as described in Materials and Methods. Means of three independent experiments are shown.

FIG. 2.

Subcellular localization of DAGAT. Homogenate (A), lipid particles (B), plasma membrane (C), vacuoles (D), 30,000 × g microsomes (E), 40,000 × g microsomes (F), 100,000 × g microsomes (G), mitochondria (H), and cytosol (I) of the wild-type strain X2180-1A were tested for DAGAT activity as described in Materials and Methods. Values are means from three independent experiments.

FIG. 3.

TAG concentration in dga1 and lro1 strains. Neutral lipids of the wild-type strain FY1679 (A) and dga1 (B), lro1 (C), and dga1 lro1 (D) strains were isolated from cells grown to the late logarithmic phase. TAGs were quantified as described in Materials and Methods, and the value for FY1679 was set at 100%. Values are means from three independent experiments.

FIG. 4.

Pathways for TAG synthesis in S. cerevisiae. FFA, free fatty acid; PC, phosphatidylcholine.