Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis

Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis

^*Gladstone Institute of Cardiovascular Disease, ^†Cardiovascular Research Institute, and ^§Department of Medicine, University of California, San Francisco, CA 94143; ^‡Veterans Administration Medical Center, San Francisco, CA 94121; ^¶Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and ^‖Departments of Microbiology and Molecular Genetics, and Medicine, and Molecular Biology Institute, University of California, Los Angeles, CA 90095

Communicated by Richard J. Havel, University of California, San Francisco, CA (received for review June 24, 1998)

Abstract

Triacylglycerols are quantitatively the most important storage form of energy for eukaryotic cells. Acyl CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the terminal and only committed step in triacylglycerol synthesis, by using diacylglycerol and fatty acyl CoA as substrates. DGAT plays a fundamental role in the metabolism of cellular diacylglycerol and is important in higher eukaryotes for physiologic processes involving triacylglycerol metabolism such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, and lactation. DGAT is an integral membrane protein that has never been purified to homogeneity, nor has its gene been cloned. We identified an expressed sequence tag clone that shared regions of similarity with acyl CoA:cholesterol acyltransferase, an enzyme that also uses fatty acyl CoA as a substrate. Expression of a mouse cDNA for this expressed sequence tag in insect cells resulted in high levels of DGAT activity in cell membranes. No other acyltransferase activity was detected when a variety of substrates, including cholesterol, were used as acyl acceptors. The gene was expressed in all tissues examined; during differentiation of NIH 3T3-L1 cells into adipocytes, its expression increased markedly in parallel with increases in DGAT activity. The identification of this cDNA encoding a DGAT will greatly facilitate studies of cellular glycerolipid metabolism and its regulation.

fatty acid/cloning/expressed sequence tag/glycerolipid

Footnotes

↵ ** Present address: Columbia University, College of Physicians and Surgeons, New York, NY, 10032
↵ ‡‡ To whom reprint requests should be addressed at: Gladstone Institute of Cardiovascular Disease, P.O. Box 419100, San Francisco, CA 94141-9100. e-mail: bfarese{at}gladstone.ucsf.edu.
Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF078752).
↵ †† Portions of this work were presented at the Experimental Biology meeting (San Francisco, 1998) and have been published in abstract form (34).
ABBREVIATIONS:

ACAT,

acyl CoA:cholesterol acyltransferase;

DAG,

diacylglycerol;

DGAT,

acyl CoA:diacylglycerol acyltransferase;

EST,

expressed sequence tag;

MAG,

monoacylglycerol;

MOI,

multiplicity of infection