Department of Molecular Medicine, Cornell University College of Veterinary Medicine, Ithaca, New York 14853, USA.
J Biol Chem. 2013 Aug 2;288(31):22862-70. doi: 10.1074/jbc.M113.458794. Epub 2013 Jun 22.
The formation of dimers or higher-order oligomers is a property of numerous integral membrane proteins, including ion channels, transporters, and receptors. In this study, we examined whether members of the DHHC-S-acyltransferase family oligomerize in intact cells and in vitro. DHHC-S-acyltransferases are integral membrane proteins that catalyze the addition of palmitate to cysteine residues on proteins at the cytoplasmic face of cell membranes. Bioluminescence resonance energy transfer (BRET) experiments revealed that DHHC2 or DHHC3 (Golgi-specific DHHC zinc finger protein (GODZ)) self-associate when expressed in HEK-293 cells. Homomultimer formation was confirmed by coimmunoprecipitation. Purified DHHC3 resolved predominately as a monomer and dimer on blue native polyacrylamide gels. In intact cells and in vitro, catalytically inactive DHHC proteins displayed a greater propensity to form dimers. BRET signals were higher for the catalytically inactive DHHC2 or DHHC3 than their wild-type counterparts. DHHC3 BRET in cell membranes was decreased by the addition of its lipid substrate palmitoyl-CoA, a treatment that results in autoacylation of the enzyme. Enzyme activity of a covalently linked DHHC3 dimer was less than that of the monomeric form, suggesting that enzyme activity may be modulated by the oligomerization status of the protein.
二聚体或更高阶寡聚体的形成是许多整合膜蛋白的特性，包括离子通道、转运蛋白和受体。在这项研究中，我们研究了 DHHC-S-酰基转移酶家族成员是否在完整细胞中和体外发生寡聚化。DHHC-S-酰基转移酶是整合膜蛋白，它们催化将棕榈酸添加到细胞质膜细胞质侧蛋白上的半胱氨酸残基上。生物发光共振能量转移 (BRET) 实验表明，DHHC2 或 DHHC3（高尔基特异性 DHHC 锌指蛋白 (GODZ)）在表达于 HEK-293 细胞时会自组装。同源多聚体形成通过共免疫沉淀得到证实。纯化的 DHHC3 在蓝色非变性聚丙烯酰胺凝胶上主要以单体和二聚体形式存在。在完整细胞中和体外，催化失活的 DHHC 蛋白显示出更强的二聚体形成倾向。与野生型相比，催化失活的 DHHC2 或 DHHC3 的 BRET 信号更高。添加其脂质底物棕榈酰-CoA 会降低细胞膜中 DHHC3 的 BRET，这一处理会导致酶的自身酰化。共价连接的 DHHC3 二聚体的酶活性小于单体形式的酶活性，这表明酶活性可能受蛋白质的寡聚状态调节。
