Fleming K G, Hohl T M, Yu R C, Müller S A, Wolpensinger B, Engel A, Engelhardt H, Brünger A T, Söllner T H, Hanson P I
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
J Biol Chem. 1998 Jun 19;273(25):15675-81. doi: 10.1074/jbc.273.25.15675.
The N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase that plays an essential role in intracellular membrane trafficking. Previous reports have concluded that NSF forms either a tetramer or a trimer in solution, and that assembly of the oligomer is essential for efficient activity in membrane transport reactions. However, in recent electron microscopic analyses NSF appears as a hexagonal cylinder similar in size to related ATPases known to be hexamers. We have therefore reevaluated NSF's oligomeric state using a variety of quantitative biophysical techniques. Sedimentation equilibrium and sedimentation velocity analytical ultracentrifugation, transmission electron microscopy with rotational image analysis, scanning transmission electron microscopy, and multiangle light scattering all demonstrate that, in the presence of nucleotide, NSF is predominantly a hexamer. Sedimentation equilibrium results further suggest that the NSF hexamer is held together by oligomerization of its D2 domains. The sedimentation coefficient, s20,w0, of 13.4 (+/-0. 1) S indicates that NSF has unusual hydrodynamic characteristics that cannot be solely explained by its shape. The demonstration that NSF is a hexameric oligomer highlights structural similarities between it and several related ATPases which act by switching the conformational states of their protein substrates in order to activate them for subsequent reactions.
N - 乙基马来酰亚胺敏感融合蛋白（NSF）是一种ATP酶，在细胞内膜运输中起关键作用。先前的报道得出结论，NSF在溶液中形成四聚体或三聚体，并且寡聚体的组装对于膜运输反应中的高效活性至关重要。然而，在最近的电子显微镜分析中，NSF呈现为六边形圆柱体，其大小与已知为六聚体的相关ATP酶相似。因此，我们使用各种定量生物物理技术重新评估了NSF的寡聚状态。沉降平衡和沉降速度分析超速离心、旋转图像分析透射电子显微镜、扫描透射电子显微镜以及多角度光散射均表明，在核苷酸存在的情况下，NSF主要是六聚体。沉降平衡结果进一步表明，NSF六聚体通过其D2结构域的寡聚化而结合在一起。13.4（±0.1）S的沉降系数s20,w0表明NSF具有不同寻常的流体动力学特性，这不能仅由其形状来解释。NSF是六聚体寡聚体的证明突出了它与几种相关ATP酶之间的结构相似性，这些ATP酶通过切换其蛋白质底物的构象状态来激活它们以进行后续反应。
