STRUCTURE AND FUNCTION OF DYNAMIN, A 100KD GTPASE INVOLVED IN ENDOCYTOSIS

参与内吞作用的 100KD GTP 酶 Dynamin 的结构和功能

• 批准号: 6105945
• 负责人: Jenny E Hinshaw
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6105945
• 关键词: Sf9 cell line; X ray crystallography; clathrin; conformation; dynamin; electron density; enzyme activity; enzyme structure; guanosinetriphosphatases; immunoelectron microscopy; receptor mediated endocytosis; sedimentation; vesicle /vacuole

Dynamin, a 100 kDa GTPase, is believed to be involved in the constriction of clathrin coated pits and may cause fission of clathrin coated vesicles during receptor mediated endocytosis and during membrane retrieval in nerve terminals. It has been shown that purified dynamin incubated under low salt conditions forms rings and spirals which, in dimension and appearance, resemble the dense material occasionally observed at the necks of coated pits. We have show that purified dynamin can also be induced to form spirals under physiological salt conditions by incubating it with GDP and g-phosphate analogues (BeF or AlF) or by dialyzing it into GTPgS. This demonstrates that the polymerized state of dynamin is markedly stabilized by sustaining its GTP or GDP/Pi-bound conformation. The formation of dynamin spirals was quantified by a sedimentation assay and imaged by electron microscopy using either negative staining techniques. Currently we are overexpressing and purifying recombinant dynamin fragments to determine which regions of dynamin are responsible for assembly. Our new ability to stabilize dynamin spirals in physiological salt solution strengthens the idea that in vivo, even in the absence of other protein cofactors, dynamin can assemble into spirals around the necks of coated pits and thereby promote vesicle fission. We have also shown by negative stain electron microscopy that purified recombinant dynamin is able to bind to lipid vesicles to form helical tubes which are similar in dimensions to dense material seen around the necks of clathrin-coated pits. By using both light and electron microscopy, we have evidence that GTP hydrolysis causes a major structural change in the dynamin/lipid tubes which may represent the process which occurs upon vesicle scission. When GTP is added to dynamin tubes they appear to constrict and form small vesicles. These results provide strong evidence that dynamin is the structural component necessary for the formation of the constricted necks of coated pits, and support the hypothesis that dynamin is the force-generating molecule responsible for membrane fission. To further understand dynamin?s role in membrane fission we are currently calculating a three-dimensional map of dynamin using cryo-electron microscopy and helical reconstruction methods. We have been able to obtain tubes that diffract to approximately ~23 angstroms which have been used to compute a preliminary 3D map. These results clearly show a lipid bilayer with dynamin molecules extending out in the shape of a ?T?. We are presently examining wild-type and mutant dynamin by this method as well as dynamin in different nucleotide states. This work will help elucidate how dynamin molecules interact and change conformation upon GTP addition and ultimately provide clues to how dynamin is regulated in the cell during endocytosis.

发动蛋白是一种100 kDa的GTdR，被认为是 参与网格蛋白包被纹孔的收缩，并可能导致 在受体介导期间网格蛋白包被的囊泡的分裂 内吞作用和在神经末梢的膜回收期间。它 已经表明，在低盐条件下孵育的纯化发动蛋白 条件形成环和螺旋，在尺寸和 外观，类似于偶尔观察到的致密物质， 被膜纹孔的颈部我们已经证明，纯化的发动蛋白可以 在生理盐条件下也可诱导形成螺旋 通过将其与GDP和g-磷酸盐类似物（BeF或AlF）一起孵育 或者通过透析将其转化为GTPgS。这表明 发动蛋白的聚合状态通过维持其 GTP或GDP/Pi结合构象。发动蛋白的形成 螺旋通过沉降试验定量，并通过 使用负染色技术进行电子显微镜检查。 目前，我们正在过度表达和纯化重组 发动蛋白片段，以确定发动蛋白的哪些区域 负责组装。我们新的稳定发动机的能力 生理盐溶液中的螺旋强化了这样的想法， 在体内，即使在没有其他蛋白质辅因子的情况下，发动蛋白可以 在被覆盖的凹坑的颈部周围组装成螺旋形， 促进囊泡分裂。 我们还通过负染色显示 电子显微镜，纯化的重组发动蛋白能够， 与脂质囊泡结合形成螺旋管， 颈部周围的致密材料尺寸 网格蛋白包被的凹坑通过使用光学和电子显微镜， 我们有证据表明GTP水解会导致一个主要的结构性变化， 发动蛋白/脂质管的变化可能代表了这一过程 这发生在囊泡破裂时。将GTP添加到dynamin时 它们似乎收缩并形成小泡。这些结果 提供了强有力的证据证明发动蛋白是 对于形成被涂覆的凹坑的收缩颈部是必要的， 并支持发动蛋白是 负责膜分裂的分子。进一步了解 发动蛋白？的作用，我们目前正在计算一个 发动蛋白冷冻电镜三维图谱 和螺旋重建方法。我们已经获得了 电子管的折射率约为~23埃， 用于计算初步的3D地图。这些结果清楚地表明 一种脂质双层，其中发动蛋白分子以 是吗？T？我们目前正在研究野生型和突变发动蛋白 以及不同核苷酸状态下的发动蛋白。 这项工作将有助于阐明发动蛋白分子如何相互作用， 在GTP添加后改变构象并最终提供 这是细胞内吞过程中发动蛋白如何调节的线索。