title: Membrane topology of human monoacylglycerol acyltransferase-2 and identification of regions important for its localization to the endoplasmic reticulum.
abstract: Acyl CoA:2-monoacylglycerol acyltransferase (MGAT)-2 has an important role in dietary fat absorption in the intestine. MGAT2 resides in the endoplasmic reticulum and catalyzes the synthesis of diacylglycerol which is then utilized as a substrate for triacylglycerol synthesis. This triacylglycerol is then incorporated into chylomicrons which are released into the circulation. In this study, we determined the membrane topology of human MGAT2. Protease protection experiments showed that the C-terminus is exposed to the cytosol, while the N-terminus is partially buried in the ER membrane. MGAT2, like murine DGAT2, was found to have two transmembrane domains. We also identified a region of MGAT2 associated with the ER membrane that contains the histidine-proline-histidine-glycine sequence present in all DGAT2 family members that is thought to comprise the active site. Proteolysis experiments demonstrated that digestion of total cellular membranes from cells expressing MGAT2 with trypsin abolished MGAT activity, indicating that domains that are important for catalysis face the cytosol. We also explored the role that the five cysteines residues present in MGAT2 have in catalysis. MGAT activity was sensitive to two thiol modifiers, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid). Furthermore, mutation of four cysteines resulted in a reduction in MGAT activity. However, when the C-terminal cysteine (C334) was mutated, MGAT activity was actually higher than that of wild-type FL-MGAT2. Lastly, we determined that both transmembrane domains of MGAT2 are important for its ER localization, and that MGAT2 is present in mitochondrial-associated membranes.
authors.affiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: McFie
authors.affiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Izzard
authors.affiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.
authors.forename: Huyen
authors.initials: H
authors.lastname: Vu
authors.affiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.
authors.forename: Youzhi
authors.initials: Y
authors.lastname: Jin
authors.affiliation: Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Erwan
authors.initials: E
authors.lastname: Beauchamp
authors.affiliation: Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Luc G
authors.initials: LG
authors.lastname: Berthiaume
authors.affiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada. Electronic address: scot.stone@usask.ca.
authors.forename: Scot J
authors.initials: SJ
authors.lastname: Stone
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004075:Diglycerides / Q000096:biosynthesis; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000201:enzymology; D050155:Lipogenesis / Q000235:genetics*; D008566:Membranes / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis / Q000235:genetics*; D014280:Triglycerides / Q000096:biosynthesis
chemical_list: D000214:Acyl Coenzyme A; D004075:Diglycerides; D014280:Triglycerides; D017351:N-Acetylglucosaminyltransferases; C063313:alpha-1,6-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase
keywords: Cysteine; Diacylglycerol; MGAT; Mitochondrial-associated membranes; Topology; Triacylglycerol