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
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
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DOI：
10.1074/jbc.m110.163691
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发表时间：
2010-11-26
影响因子：
4.8
通讯作者：
Stone, Scot J.
中科院分区：
文献类型：
--
作者：
McFie, Pamela J.;Stone, Sandra L.;Stone, Scot J.
酰基CoA：二酰基甘油酰基转移酶（DGAT）是催化三酰基甘油合成的内质网的整合膜蛋白。已经鉴定了两种DGAT酶（DGAT 1和DGAT 2），其在脂质代谢中具有独特的作用。DGAT 1是一种多功能酰基转移酶，除了三酰基甘油之外，还能够合成二酰基甘油、视黄基和蜡酯。在这里，我们报告的膜拓扑结构的小鼠DGAT 1蛋白酶保护试验和间接免疫荧光结合选择性透化的细胞膜。基于预测算法的拓扑结构模型表明DGAT 1具有8个跨膜结构域。与此相反，我们的数据表明，DGAT 1有三个跨膜结构域的N端朝向胞质溶胶。DGAT 1的C-末端区域，占蛋白质的50%左右，存在于内质网腔中，并含有高度保守的组氨酸残基（His-426），可能是活性位点的一部分。His-426突变为丙氨酸，在体外酰基转移酶测定中损害DGAT 1合成三酰甘油以及视黄基酯和蜡酯的能力。最后，我们表明，N-末端结构域的DGAT 1的催化活性是不需要的DGAT 1，但相反，可能参与调节酶的活性和二聚体/四聚体的形成。
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 similar to 50% 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.