Hirst Jennifer, Sahlender Daniela A, Li Sam, Lubben Nienke B, Borner Georg H H, Robinson Margaret S
Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust, Addenbrooke's Hospital, Cambridge, UK.
Traffic. 2008 Aug;9(8):1354-71. doi: 10.1111/j.1600-0854.2008.00764.x. Epub 2008 May 17.
Auxilin is a cofactor for Hsc70-mediated uncoating of clathrin-coated vesicles (CCVs). However, small interfering RNA (siRNA) knockdown of the ubiquitous auxilin 2 in HeLa cells only moderately impairs clathrin-dependent trafficking. In this study, we show that HeLa cells also express auxilin 1, previously thought to be neuron specific, and that both auxilins need to be depleted for inhibition of clathrin-mediated endocytosis and intracellular sorting. Depleting both auxilins cause an approximately 50% reduction in the number of clathrin-coated pits at the plasma membrane but enhances the association of clathrin and adaptors with intracellular membranes. CCV fractions isolated from auxilin-depleted cells have an approximately 1.5-fold increase in clathrin content and more than fivefold increase in the amount of AP-2 adaptor complex and other endocytic machinery, with no concomitant increase in cargo. In addition, the structures isolated from auxilin-depleted cells are on average smaller than CCVs from control cells and are largely devoid of membrane, indicating that they are not CCVs but membraneless clathrin cages. Similar structures are observed by electron microscopy in intact auxilin-depleted HeLa cells. Together, these findings indicate that the two auxilins have overlapping functions and that they not only facilitate the uncoating of CCVs but also prevent the formation of nonproductive clathrin cages in the cytosol.
辅助蛋白是Hsc70介导的网格蛋白包被小泡（CCV）脱包被过程的辅助因子。然而，在HeLa细胞中对普遍存在的辅助蛋白2进行小干扰RNA（siRNA）敲低，仅适度损害网格蛋白依赖性运输。在本研究中，我们发现HeLa细胞还表达辅助蛋白1（以前认为是神经元特异性的），并且两种辅助蛋白都需要被耗尽才能抑制网格蛋白介导的内吞作用和细胞内分选。耗尽这两种辅助蛋白会导致质膜上网格蛋白包被小窝的数量减少约50%，但会增强网格蛋白和衔接蛋白与细胞内膜的结合。从辅助蛋白耗尽的细胞中分离出的CCV组分，其网格蛋白含量增加约1.5倍，AP-2衔接蛋白复合物和其他内吞机制的量增加超过五倍，而货物没有相应增加。此外，从辅助蛋白耗尽的细胞中分离出的结构平均比对照细胞的CCV小，并且基本上没有膜，这表明它们不是CCV，而是无膜的网格蛋白笼。在完整的辅助蛋白耗尽的HeLa细胞中通过电子显微镜观察到类似的结构。总之，这些发现表明这两种辅助蛋白具有重叠的功能，并且它们不仅促进CCV的脱包被，还防止在细胞质中形成无生产性的网格蛋白笼。
