title: Human acyl-CoA:diacylglycerol acyltransferase is a tetrameric protein.
abstract: Acyl-CoA:diacylglycerol acyltransferase (DGAT) is an integral membrane enzyme that catalyses the last step of triacylglycerol synthesis from diacylglycerol and acyl-CoA. Here we provide experimental evidence that DGAT is a homotetramer. Although the predicted molecular mass of human DGAT protein is 55 kDa, CHAPS-solubilized recombinant human DGAT was eluted in fractions over 150 kDa on gel-filtration chromatography. Cross-linking of recombinant DGAT in membranes with disuccinimidyl suberate yielded bands corresponding to the dimer (108 kDa) and the tetramer (214 kDa) in SDS/PAGE. Finally, when two differently epitope-tagged forms of DGAT were co-transfected into mammalian cells, they could be co-immunoprecipitated. From a human adipose tissue cDNA library we cloned a cDNA encoding a novel splice variant of DGAT (designated DGATsv) that contained a 77 nt insert of unspliced intron with an in-frame stop codon. This resulted in a truncated form of DGAT that terminated at Arg-387, deleting 101 residues from the C-terminus containing the putative active site. DGATsv was enzymically inactive when transfected in HEK-293E cells but was still able to form dimer and tetramer on cross-linking, indicating that the ability to form tetramers resides in the N-terminal region. When co-expressed in HEK-293E cells, DGATsv did not inhibit the activity of full-length DGAT, suggesting that the subunits of DGAT catalyse triacylglycerol synthesis independently.
authors.affiliation: Department of Metabolic Diseases, DuPont Pharmaceuticals Company, Experimental Station, Route 141 and Henry Clay Road, Wilmington, DE 19880-0400, USA. dong.cheng@dupontpharma.com
authors.forename: D
authors.initials: D
authors.lastname: Cheng
authors.forename: R L
authors.initials: RL
authors.lastname: Meegalla
authors.forename: B
authors.initials: B
authors.lastname: He
authors.forename: D A
authors.initials: DA
authors.lastname: Cromley
authors.forename: J T
authors.initials: JT
authors.lastname: Billheimer
authors.forename: P R
authors.initials: PR
authors.lastname: Young
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D002460:Cell Line; D002793:Cholic Acids / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D051048:Diacylglycerol O-Acyltransferase; D017930:Genes, Reporter; D006801:Humans; D008970:Molecular Weight; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D002793:Cholic Acids; D003902:Detergents; D011993:Recombinant Fusion Proteins; D000217:Acyltransferases; C494881:DGAT1 protein, human; D051048:Diacylglycerol O-Acyltransferase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate