Whiteheart S W, Rossnagel K, Buhrow S A, Brunner M, Jaenicke R, Rothman J E
Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York 10021.
J Cell Biol. 1994 Aug;126(4):945-54. doi: 10.1083/jcb.126.4.945.
The NEM-sensitive fusion protein, NSF, together with SNAPs (soluble NSF attachment proteins) and the SNAREs (SNAP receptors), is thought to be generally used for the fusion of transport vesicles to their target membranes. NSF is a homotrimer whose polypeptide subunits are made up of three distinct domains: an amino-terminal domain (N) and two homologous ATP-binding domains (D1 and D2). Mutants of NSF were produced in which either the order or composition of the three domains were altered. These mutants could not support intra-Golgi transport, but they indicated that the D2 domain was required for trimerization of the NSF subunits. Mutations of the first ATP-binding site that affected either the binding (K266A) or hydrolysis (E329Q) of ATP completely eliminated NSF activity. The hydrolysis mutant was an effective, reversible inhibitor of Golgi transport with an IC50 of 125 ng/50 microliters assay. Mutants in the second ATP-binding site (binding, K549A; hydrolysis, D604Q) had either 14 or 42% the specific activity of the wild-type protein, respectively. Using coexpression of an inactive mutant with wild-type subunits, it was possible to produce a recombinant form of trimeric NSF that contained a mixture of subunits. The mixed NSF trimers were inactive, even when only one mutant subunit was present, suggesting that NSF action requires each of the three subunits in a concerted mechanism. These studies demonstrate that the ability of the D1 domain to hydrolyze ATP is required for NSF activity and, therefore is required for membrane fusion. The D2 domain is required for trimerization, but its ability to hydrolyze ATP is not absolutely required for NSF function.
对N - 乙基马来酰亚胺（NEM）敏感的融合蛋白N - 乙基马来酰亚胺敏感因子（NSF），与可溶性NSF附着蛋白（SNAPs）和SNAP受体（SNAREs）一起，被认为普遍用于运输小泡与靶膜的融合。NSF是一种同三聚体，其多肽亚基由三个不同的结构域组成：一个氨基末端结构域（N）和两个同源的ATP结合结构域（D1和D2）。构建了NSF的突变体，其中三个结构域的顺序或组成发生了改变。这些突变体不能支持高尔基体内部的运输，但它们表明D2结构域是NSF亚基三聚化所必需的。影响ATP结合（K266A）或水解（E329Q）的第一个ATP结合位点的突变完全消除了NSF活性。水解突变体是高尔基体运输的有效、可逆抑制剂，在50微升测定中IC50为125纳克。第二个ATP结合位点的突变体（结合，K549A；水解，D604Q）分别具有野生型蛋白比活性的14%或42%。通过将无活性突变体与野生型亚基共表达，可以产生包含亚基混合物的三聚体NSF的重组形式。即使只存在一个突变亚基，混合的NSF三聚体也是无活性的，这表明NSF的作用需要三个亚基协同作用。这些研究表明，D1结构域水解ATP的能力是NSF活性所必需的，因此也是膜融合所必需的。D2结构域是三聚化所必需的，但它水解ATP的能力对于NSF功能不是绝对必需的。
