Murine Diacylglycerol Acyltransferase-2 (DGAT2) Can Catalyze Triacylglycerol Synthesis and Promote Lipid Droplet Formation Independent of Its Localization to the Endoplasmic Reticulum

Abstract

Triacylglycerol (TG) is the major form of stored energy in eukaryotic organisms and is synthesized by two distinct acylCoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2. Both DGAT enzymes reside in the endoplasmic reticulum (ER), but DGAT2 also co-localizes with mitochondria and lipid droplets. In this report, we demonstrate that murine DGAT2 is part of a multimeric complex consisting of several DGAT2 subunits. We also identified the region of DGAT2 responsible for its localization to the ER. A DGAT2 mutant lacking both its transmembrane domains, although still associated with membranes, was absent from the ER and instead localized to mitochondria. Unexpectedly, this mutant was still active and capable of interacting with lipid droplets to promote TG storage. Additional experiments indicated that the ER targeting signal was present in the first transmembrane domain (TMD1) of DGAT2. When fused to a fluorescent reporter, TMD1, but not TMD2, was sufficient to target mCherry to the ER. Finally, the interaction of DGAT2 with lipid droplets was dependent on the C terminus of DGAT2. DGAT2 mutants, in which regions of the C terminus were either truncated or specific regions were deleted, failed to co-localize with lipid droplets when cells were oleate loaded to stimulate TG synthesis. Our findings demonstrate that DGAT2 is capable of catalyzing TG synthesis and promote its storage in cytosolic lipid droplets independent of its localization in the ER. Triacylglycerols (TG),2 the major form of stored energy in eukaryotic organisms, are synthesized via the multistep glycerol 3-phosphate or Kennedy pathway (1). In this pathway, three fatty acyl-coenzyme A (CoA) molecules (activated forms of fatty acids) are linked via ester bonds to a glycerol backbone. The microsomal enzyme, acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the final reaction of the pathway by forming an ester bond between a long chain fatty acid and the free hydroxyl group of diacylglycerol (DG) generating TG.Two different DGAT enzymes, DGAT1 and DGAT2, are responsible for the acyl-CoA-dependent synthesis of TG in mammalian tissues (2-4). The genes encoding these two enzymes have no sequence homology and they each belong to distinct gene families. DGAT1 belongs to a large family of membrane-bound O-acyltransferases that includes acyl-CoA: cholesterol acyltransferase (ACAT)-1 and -2, which catalyze cholesterol ester biosynthesis (5-9). DGAT2 belongs to the DGAT2/acyl-CoA:monoacylglycerol acyltransferase gene family that includes monoacylglycerol acyl-CoA acyltransferases 1-3 and wax synthase (3, 8, 10 -14). In mice and humans, DGAT1 and DGAT2 are expressed in most tissues, with the highest levels of expression found in tissues that are associated with TG metabolism (adipose tissue, liver, small intestine, and mammary gland) (3, 5).Studies in mice and in cells in culture have provided compelling evidence that DGAT2, more so than DGAT1, is responsible for the majority of TG synthesis and that these two enzymes have separa...

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