School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK.
Traffic. 2013 Sep;14(9):987-96. doi: 10.1111/tra.12085. Epub 2013 Jun 20.
The molecular chaperone, Hsc70, together with its co-factor, auxilin, facilitates the ATP-dependent removal of clathrin during clathrin-mediated endocytosis in cells. We have used cryo-electron microscopy to determine the 3D structure of a complex of clathrin, auxilin(401-910) and Hsc70 at pH 6 in the presence of ATP, frozen within 20 seconds of adding Hsc70 in order to visualize events that follow the binding of Hsc70 to clathrin and auxilin before clathrin disassembly. In this map, we observe density beneath the vertex of the cage that we attribute to bound Hsc70. This density emerges asymmetrically from the clathrin vertex, suggesting preferential binding by Hsc70 for one of the three possible sites at the vertex. Statistical comparison with a map of whole auxilin and clathrin previously published by us reveals the location of statistically significant differences which implicate involvement of clathrin light chains in structural rearrangements which occur after Hsc70 is recruited. Clathrin disassembly assays using light scattering suggest that loss of clathrin light chains reduces the efficiency with which auxilin facilitates this reaction. These data support a regulatory role for clathrin light chains in clathrin disassembly in addition to their established role in regulating clathrin assembly.
分子伴侣 Hsc70 与其辅助因子 auxilin 一起促进了网格蛋白介导的内吞作用中网格蛋白的 ATP 依赖性去除。我们使用冷冻电子显微镜在 pH6 下、存在 ATP 的条件下，确定了网格蛋白、auxilin(401-910)和 Hsc70 的复合物的 3D 结构，在添加 Hsc70 后 20 秒内将其冷冻，以便可视化 Hsc70 与网格蛋白和 auxilin 结合后、网格蛋白解组装之前发生的事件。在这个图谱中，我们观察到笼状结构顶点下方的密度，我们将其归因于结合的 Hsc70。这种密度从网格蛋白顶点不对称地出现，表明 Hsc70 优先结合顶点三个可能的位点之一。与我们之前发表的整个 auxilin 和网格蛋白图谱的统计比较揭示了位置的显著差异，这暗示了网格蛋白轻链参与了 Hsc70 被招募后发生的结构重排。使用光散射的网格蛋白解组装测定表明，网格蛋白轻链的丢失降低了 auxilin 促进该反应的效率。这些数据支持网格蛋白轻链在网格蛋白解组装中的调节作用，除了它们在调节网格蛋白组装中的既定作用之外。
