EpsinR is an adaptor for the SNARE protein Vti1b
EpsinR is an adaptor for the SNARE protein Vti1b
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DOI：
10.1091/mbc.e04-06-0468
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发表时间：
2004-12-01
影响因子：
3.3
通讯作者：
Robinson, MS
中科院分区：
文献类型：
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作者：
Hirst, J;Miller, SE;Robinson, MS
EpsinR是一种与vti1b结合的笼蛋白涂层囊泡(CCV)相关蛋白，提示它可能是一个vti1b选择性接头。在HeLa细胞中，使用siRNA将epsinR耗尽到无法检测到的水平会导致vti1b从核周区重新分布到细胞外围，但vti1a也在epsinR耗竭的细胞中重新分布，这两种VTI亚型都在AP-1耗竭的细胞中重新分布。为了更直接地测试epsinR的功能，我们从对照和siRNA处理的细胞中分离出CCV，然后寻找货物含量的差异。在网状蛋白缺失的细胞中，外壳蛋白和货运蛋白在这种制备过程中都大大减少。敲除epsinR会导致AP-1与CCV共分泌的数量减少50%，反之亦然，这表明这两种蛋白质相互依赖，才能最大限度地结合到外壳中。此外，在来自epsinR和AP-1耗竭细胞的CCV中，vti1b而不是vti1a减少了70%。由于AP-1基因敲除减少了CCV中epsinR的数量，因此其对vti1b的影响可能是间接的。这些发现提供了体内证据，表明epsinR是vti1b的接头，也表明CCV分离可用于接头功能的检测。
EpsinR is a clathrin-coated vesicle (CCV)-associated protein that binds to vti1b, suggesting that it may be a vti1b-selective adaptor. Depletion of epsinR to undetectable levels in HeLa cells using siRNA causes vti1b to redistribute from the perinuclear region to the cell periphery, but vti1a also redistributes in epsinR-depleted cells, and both vti isoforms redistribute in AP-1-depleted cells. As a more direct assay for epsinR function, we isolated CCVs from control and siRNA-treated cells and then looked for differences in cargo content. In clathrin-depleted cells, both coat and cargo proteins are greatly reduced in this preparation. Knocking down epsinR causes a similar to50% reduction in the amount of AP-1 copurifying with CCVs and vice versa, indicating that the two proteins are dependent on each other for maximum incorporation into the coat. In addition, vti1b, but not vti1a, is reduced by >70% in CCVs from both epsinR- and AP-1-depleted cells. Because AP-1 knockdown reduces the amount of epsinR in CCVs, it is possible that its effect on vti1b may be indirect. These findings provide in vivo evidence that epsinR is an adaptor for vti1b, and they also show that CCV isolation can be used as an assay for adaptor function.