Multiple distinct coiled-coils are involved in dynamin self-assembly
Multiple distinct coiled-coils are involved in dynamin self-assembly
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DOI：
10.1074/jbc.274.15.10277
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发表时间：
1999-04-09
影响因子：
4.8
通讯作者：
Vallee, RB
中科院分区：
文献类型：
--
作者：
Okamoto, PM;Tripet, B;Vallee, RB
Dynamin是一种100 kDa的GTP酶，参与了突触小泡循环、受体介导的内吞作用和其他膜分离过程。动力素在被涂覆的凹坑和其他膜凹陷的颈部自组装成螺旋环，并介导膜的断裂。在体外，动力蛋白已被报道以二聚体、四聚体、环状低聚物和螺旋聚合物的形式存在。在这项研究中，我们试图定义Dynamin中的自组装区，通过共免疫沉淀和酵母相互作用陷阱检测到Dynamin-1C末端附近两个紧密间隔的序列的缺失，取消了自结合，并将沉降系数从7.5%降低到4.5%。S，合成多肽的圆二色谱和平衡超速离心法表明，在C端组装结构域和第三个中心位置处形成了卷曲的螺旋。其中两个多肽形成了四聚体，支持每个多肽在单体-四聚体转变中的作用，并为四聚体的组织提供了新的见解，部分缺失的C末端组装结构域逆转了Dynamin-1 GTP酶突变体对内吞作用的主要抑制。不同的动力蛋白异构体之间也观察到了自结合，我们的结果揭示了两个不同的包含螺旋线圈的组装结构域，它们可以识别其他动力蛋白异构体并介导内吞抑制。此外，我们的数据有力地表明了动力蛋白亚单位自关联的平行模型。
Dynamin, a 100-kDa GTPase, has been implicated to be involved in synaptic vesicle recycling, receptor-mediated endocytosis, and other membrane sorting processes. Dynamin self-assembles into helical collars around the necks of coated pits and other membrane invaginations and mediates membrane scission. In vitro, dynamin has been reported to exist as dimers, tetramers, ring-shaped oligomers, and helical polymers. In this study we sought to define self-assembly regions in dynamin, Deletion of two closely spaced sequences near the dynamin-1 C terminus abolished self-association as assayed by co-immunoprecipitation and the yeast interaction trap, and reduced the sedimentation coefficient from 7.5 to 4.5 S, Circular dichroism spectroscopy and equilibrium ultracentrifugation of synthetic peptides revealed coiled-coil formation within the C-terminal assembly domain and at a third, centrally located site. Two of the peptides formed tetramers, supporting a role for each in the monomer-tetramer transition and providing novel insight into the organization of the tetramer, Partial deletions of the C-terminal assembly domain reversed the dominant inhibition of endocytosis by dynamin-1 GTPase mutants. Self association was also observed between different dynamin isoforms, Taken altogether, our results reveal two distinct coiled-coil-containing assembly domains that can recognize other dynamin isoforms and mediate endocytic inhibition. In addition, our data strongly suggests a parallel model for dynamin subunit self-association.