Evidence for Triacylglycerol Synthesis in the Lumen of Microsomes via a Lipolysis-Esterification Pathway Involving Carnitine Acyltransferases

Abo-Hashema, Khaled A.H.; Cake, Max H.; Power, Glen W.; Clarke, D. G.

doi:10.1074/jbc.274.50.35577

Cited by 56 publications

(47 citation statements)

References 53 publications

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“…This model agrees with previous biochemical studies in which the active site of DGAT (and that of the family member acyl-CoA:monoacylglycerol acyltransferase) was oriented toward the cytosol (24). A latent DGAT activity (present only after treating membranes with disrupting agents) has been detected in rat liver microsomes (25)(26)(27)(28), suggesting that there are active sites on both sides of the ER membrane. Our topology data suggest that DGAT2 is not responsible for this latent activity.…”

Section: Discussionsupporting

confidence: 81%

Membrane Topology and Identification of Key Functional Amino Acid Residues of Murine Acyl-CoA:Diacylglycerol Acyltransferase-2

Stone

Levin

Farese

2006

Journal of Biological Chemistry

200

211

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Triacylglycerols are the predominant molecules of energy storage in eukaryotes. However, excessive accumulation of triacylglycerols in adipose tissue leads to obesity and, in nonadipose tissues, is associated with tissue dysfunction. Hence, it is of great importance to have a better understanding of the molecular mechanisms of triacylglycerol synthesis. The final step in triacylglycerol synthesis is catalyzed by the acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2. Although recent studies have shed light on metabolic functions of these enzymes, little is known about the molecular aspects of their structures or functions. Here we report the topology for murine DGAT2 and the identification of key amino acids that likely contribute to enzymatic function. Our data indicate that DGAT2 is an integral membrane protein with both the N and C termini oriented toward the cytosol. A long hydrophobic region spanning amino acids 66 -115 likely comprises two transmembrane domains or, alternatively, a single domain that is embedded in the membrane bilayer. The bulk of the protein lies distal to the transmembrane domains. This region shares the highest degree of homology with other enzymes of the DGAT2 family and contains a sequence HPHG that is conserved in all family members. Mutagenesis of this sequence in DGAT2 demonstrated that it is required for full enzymatic function. Additionally, a neutral lipid-binding domain that is located in the putative first transmembrane domain was also required for full enzymatic function. Our findings provide the first insights into the topography and molecular aspects of DGAT2 and related enzymes.

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Section: Discussionsupporting

confidence: 81%

Membrane Topology and Identification of Key Functional Amino Acid Residues of Murine Acyl-CoA:Diacylglycerol Acyltransferase-2

Stone

Levin

Farese

2006

Journal of Biological Chemistry

200

211

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“…A carnitine acyltransferase system capable of transporting fatty acids from the cytosol across membranes via a fatty acylcarnitine intermediate has been identified at the ER (41)(42)(43). Carnitine acyltransferase enzymes have previously been demonstrated to have a role in TG synthesis in the ER lumen both in vivo in rat small intestine and with a cell-free system, providing evidence for a mechanism that could deliver fatty acid substrates across the ER membrane from the cytosol to the active site of DGAT1 in the ER lumen (44,45).…”

Section: Discussionmentioning

confidence: 99%

Topological Orientation of Acyl-CoA:Diacylglycerol Acyltransferase-1 (DGAT1) and Identification of a Putative Active Site Histidine and the Role of the N Terminus in Dimer/Tetramer Formation

McFie

Stone

Banman

et al. 2010

Journal of Biological Chemistry

133

125

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Acyl CoA:diacylglycerol acyltransferase (DGAT) is an integral membrane protein of the endoplasmic reticulum that catalyzes the synthesis of triacylglycerols. Two DGAT enzymes have been identified (DGAT1 and DGAT2) with unique roles in lipid metabolism. DGAT1 is a multifunctional acyltransferase capable of synthesizing diacylglycerol, retinyl, and wax esters in addition to triacylglycerol. Here, we report the membrane topology for murine DGAT1 using protease protections assays and indirect immunofluorescence in conjunction with selective permeabilization of cellular membranes. Topology models based on prediction algorithms suggested that DGAT1 had eight transmembrane domains. In contrast, our data indicate that DGAT1 has three transmembrane domains with the N terminus oriented toward the cytosol. The C-terminal region of DGAT1, which accounts for ϳ50% of the protein, is present in the endoplasmic reticulum lumen and contains a highly conserved histidine residue (His-426) that may be part of the active site. Mutagenesis of His-426 to alanine impaired the ability of DGAT1 to synthesize triacylglycerols as well as retinyl and wax esters in an in vitro acyltransferase assay. Finally, we show that the N-terminal domain of DGAT1 is not required for the catalytic activity of DGAT1 but, instead, may be involved in regulating enzyme activity and dimer/tetramer formation. Acyl coenzyme A:1,2-diacylglycerol acyltransferase (DGAT)2 is a membrane-bound enzyme that catalyzes the biosynthesis of triacylglycerols (TGs) (1). TGs are a class of neutral lipid that represents the major form of stored energy in eukaryotic organisms (2). However, excessive accumulation of TG in tissues can lead to obesity and insulin resistance, whereas increased TG in the blood is a risk factor for atherosclerosis (3)(4)(5).DGAT catalyzes the formation of an ester bond between a long chain fatty acid (fatty acyl coenzyme A (CoA)) and the free hydroxyl group of diacylglycerol (DG), generating TG. Two DGAT genes have now been identified, Dgat1 and Dgat2, which share no sequence homology. DGAT1 belongs to a large family of membrane-bound O-acyltransferases (MBOAT) that includes acyl-CoA:cholesterol acyltransferase-1 and -2 (ACAT-1 and -2), which catalyze cholesterol ester biosynthesis (6 -10). DGAT2 belongs to the DGAT2/acyl-CoA:monoacylglycerol acyltransferase gene family including monoacylglycerol acylCoA acyltransferases 1-3 and wax synthase (9, 11-16). Biochemical analyses have indicated that DGAT1 and DGAT2 have distinct roles in TG metabolism. When overexpressed in cells, DGAT2 yielded a much larger increase in intracellular triacylglycerol than DGAT1 (17). In in vitro assays, DGAT1, but not DGAT2, was capable of using a broad array of acyl acceptors to synthesize diacylglycerol, retinyl, and wax esters in addition to triacylglycerol (18).In vivo experiments in mice have also provided strong evidence that DGAT1 and DGAT2 do not serve redundant roles in lipid metabolism. Dgat1-deficient (Dgat1 Ϫ/Ϫ ) mice were viable with modest reductions in TG con...

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“…Two DGAT activities are found in microsomes: DGAT I or overt, cytosol-accessible DGAT, which is suggested to catalyse the synthesis of TAG destined for cytoplasmic droplets; and DGAT II, accessible to its substrates from the lumenal aspect of the endoplasmic reticular membrane 1 and thought to catalyse the synthesis of TAG on the lumenal aspect, from where it is incorporated into VLDL during the two-step process of particle lipidation. Experimental evidence for the incorporation and transfer of DAG and acyl moieites across the membrane for use by DGAT II was subsequently provided in Abo-Hashema et al 2 Consequently, the relative activities of the two DGATs may have a significant impact upon the level of triglyceridaemia, as well as on the development of steatosis. More recently, the cDNAs of genes coding for two separate DGATs have been cloned and sequenced.…”

Section: Introductionmentioning

confidence: 99%

Activities of overt and latent diacylglycerol acyltransferases (DGATs I and II) in liver microsomes of ob/ob mice

Waterman

Zammit

2002

Int J Obes

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The effect of the ob=ob mutation on microsomal overt and latent acyl CoA:diacylglycerol acyl transferase activities (DGATs I and II) in liver microsomes of mice was investigated. Overt and latent hepatic DGAT activities for 16-week-old lean Ob=? mice were 0.42 AE 0.14 and 1.57 AE 0.04 mmol=mg=min, respectively. For ob=ob mice DGAT I and II activities were 1.17 AE 0.28 and 3.09 AE 0.78 mmol=mg=min, respectively. The hepatic triacylglycerol (TAG) concentration of 16-week-old mice was increased three-fold in ob=ob mice relative to those in lean controls. The data suggest that the increased rate of hepatic TAG secretion and intracellular accumulation in ob=ob mice is accompanied by parallel increases in the activities of both DGATs.

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Evidence for Triacylglycerol Synthesis in the Lumen of Microsomes via a Lipolysis-Esterification Pathway Involving Carnitine Acyltransferases

Cited by 56 publications

References 53 publications

Membrane Topology and Identification of Key Functional Amino Acid Residues of Murine Acyl-CoA:Diacylglycerol Acyltransferase-2

Membrane Topology and Identification of Key Functional Amino Acid Residues of Murine Acyl-CoA:Diacylglycerol Acyltransferase-2

Topological Orientation of Acyl-CoA:Diacylglycerol Acyltransferase-1 (DGAT1) and Identification of a Putative Active Site Histidine and the Role of the N Terminus in Dimer/Tetramer Formation

Activities of overt and latent diacylglycerol acyltransferases (DGATs I and II) in liver microsomes of ob/ob mice

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