Dynamin assembles into spirals under physiological salt conditions upon the addition of GDP and gamma-phosphate analogues
Dynamin assembles into spirals under physiological salt conditions upon the addition of GDP and gamma-phosphate analogues
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DOI：
10.1074/jbc.272.44.28030
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发表时间：
1997-10-31
影响因子：
4.8
通讯作者：
Hinshaw, JE
中科院分区：
文献类型：
--
作者：
Carr, JF;Hinshaw, JE
发动蛋白是一种100-kDa的GTdR，据信其在受体介导的内吞作用期间和神经末梢中的膜回收期间参与网格蛋白包被的小凹的收缩和网格蛋白包被的囊泡的分裂。已经表明，在低盐条件下孵育的纯化的发动蛋白形成环和螺旋，其在尺寸和外观上类似于偶尔在涂覆的凹坑的颈部处观察到的致密材料。在这份报告中，我们表明，纯化的发动蛋白形式螺旋在生理盐条件下孵育GDP和γ-磷酸类似物（铍和氟化铝）或透析成鸟苷5 '-3-O-（硫代）三磷酸。此外，当发动蛋白被蛋白水解成主要的类似于90-kDa的物质，缺乏C末端，或两个较小的片段，一个类似于55-kDa的物质来源于N-末端的蛋白质的一半和一个类似于30-kDa的物质缺乏N和C末端时，仍然形成螺旋。这项工作表明，除了GDP和γ-磷酸类似物逮捕动力蛋白在GTP或过渡态，显着稳定的螺旋构象在生理离子强度条件下，从而表明，在没有受体的情况下，动力蛋白能够组装成螺旋在颈部的包被坑囊泡分裂之前。
Dynamin is a 100-kDa GTPase that is believed to be involved in the constriction of clathrin-coated pits and the fission of clathrin-coated vesicles during receptor-mediated endocytosis and during membrane retrieval in nerve termini. It has been shown that purified dynamin incubated under low salt conditions forms rings and spirals that, in dimension and appearance, resemble the dense material occasionally observed at the necks of coated pits. In this report we show that purified dynamin forms spirals under physiological salt conditions when incubated with GDP and gamma-phosphate analogues (beryllium and aluminum fluoride) or when dialyzed into guanosine 5'-3-O-(thio)triphosphate. Moreover, spirals still form when dynamin is proteolyzed to either a predominant similar to 90-kDa species, lacking the C terminus, or to two smaller fragments, a similar to 55-kDa species originating from the N-terminal half of the protein and a similar to 30-kDa species lacking both the N and C termini. This work indicates that the addition of GDP and gamma-phosphate analogues arrests dynamin in a GTP or transition state that markedly stabilizes the spiral conformation under physiological ionic strength conditions and thereby suggests that dynamin in the absence of a receptor is capable of assembly into spirals at the necks of coated pits prior to vesicle fission.