Ma Yuchen, Greener Tsvika, Pacold Michael E, Kaushal Shivani, Greene Lois E, Eisenberg Evan
Laboratory of Cell Biology, NHLBI/National Institutes of Health, 50 South Drive, Bethesda, MD 20892-0301, USA.
J Biol Chem. 2002 Dec 20;277(51):49267-74. doi: 10.1074/jbc.M203695200. Epub 2002 Oct 10.
During clathrin-mediated endocytosis Hsc70, supported by the J-domain protein auxilin, uncoats clathrin-coated vesicles. Auxilin contains both a clathrin-binding domain and a J-domain that binds Hsc70, and it has been suggested that these two domains are both necessary and sufficient for auxilin activity. To test this hypothesis, we created a chimeric protein consisting of the J-domain of auxilin linked to the clathrin-binding domain of the assembly protein AP180. This chimera supported uncoating, but unlike auxilin it acted stoichiometrically rather than catalytically because, like Hsc70, it remained associated with the uncoated clathrin. This observation supports our proposal that Hsc70 chaperones uncoated clathrin by inducing formation of a stable Hsc70-clathrin-AP complex. It also shows that Hsc70 acts by dissociating individual clathrin triskelions rather than cooperatively destabilizing clathrin-coated vesicles. Because the chimera lacks the C-terminal subdomain of the auxilin clathrin-binding domain, it seemed possible that this subdomain is required for auxilin to act catalytically, and indeed its deletion caused auxilin to act stoichiometrically. In contrast, deletion of the N-terminal subdomain weakened auxilin-clathrin binding and prevented auxilin from polymerizing clathrin. Therefore the C-terminal subdomain of the clathrin-binding domain of auxilin is required for auxilin to act catalytically, whereas the N-terminal subdomain strengthens auxilin-clathrin binding.
在网格蛋白介导的内吞作用中，热休克同源蛋白70（Hsc70）在J结构域蛋白辅助蛋白的支持下，使网格蛋白包被的囊泡脱包被。辅助蛋白既包含一个网格蛋白结合结构域，又包含一个与Hsc70结合的J结构域，有人提出这两个结构域对于辅助蛋白的活性而言都是必需且充分的。为了验证这一假说，我们构建了一种嵌合蛋白，该蛋白由辅助蛋白的J结构域与装配蛋白AP180的网格蛋白结合结构域相连组成。这种嵌合体支持脱包被，但与辅助蛋白不同的是，它以化学计量方式起作用而非催化作用，因为与Hsc70一样，它仍与脱包被的网格蛋白结合。这一观察结果支持了我们的提议，即Hsc70通过诱导形成稳定的Hsc70 - 网格蛋白 - AP复合物来陪伴脱包被的网格蛋白。它还表明，Hsc70的作用方式是解离单个网格蛋白三脚复合体，而不是协同破坏网格蛋白包被的囊泡的稳定性。由于该嵌合体缺少辅助蛋白网格蛋白结合结构域的C末端亚结构域，因此似乎这个亚结构域是辅助蛋白发挥催化作用所必需的，实际上其缺失导致辅助蛋白以化学计量方式起作用。相反，N末端亚结构域的缺失削弱了辅助蛋白与网格蛋白的结合，并阻止辅助蛋白使网格蛋白聚合。因此，辅助蛋白网格蛋白结合结构域的C末端亚结构域是辅助蛋白发挥催化作用所必需的，而N末端亚结构域则增强了辅助蛋白与网格蛋白的结合。
