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 55kDa, CHAPS-solubilized recombinant human DGAT was eluted in fractions over 150kDa on gel-filtration chromatography. Cross-linking of recombinant DGAT in membranes with disuccinimidyl suberate yielded bands corresponding to the dimer (108kDa) and the tetramer (214kDa) 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 77nt 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.
Human acyl-CoA:diacylglycerol acyltransferase is a tetrameric protein
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Dong CHENG, Rupalie L. MEEGALLA, Bokang HE, Debra A. CROMLEY, Jeffery T. BILLHEIMER, Peter R. YOUNG; Human acyl-CoA:diacylglycerol acyltransferase is a tetrameric protein. Biochem J 1 November 2001; 359 (3): 707–714. doi: https://doi.org/10.1042/bj3590707
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