Degradation of Mislocalized Secretory and Membrane Proteins

错误定位的分泌蛋白和膜蛋白的选择性识别和降解已在体外重建。在这个系统中，分泌和膜蛋白的合成处于非易位状态，只需从反应中省略内质网衍生的粗微体，或者如果包括它们，则通过使用辅转素抑制易位来合成。在这两种操作中，非转位蛋白被迅速泛素化，为其降解做准备。我们正在使用经典的分离和亲和纯化方法来鉴定选择性识别和降解这些非转位蛋白的机制。在过去的一年里，我们成功地在体外重组了许多这样的事件，并使用这个系统来识别因素。我们已经确定了参与其中的E2酶，以及促进降解的关键伴侣蛋白。我们目前的目标是确定这一途径的剩余因素，并用纯化的成分重建事件。这将为从机械上理解这条通路是如何运行的铺平了道路。与此同时，我们正在进入体内系统，以了解这种降解途径在维持细胞蛋白质动态平衡方面的重要性。

The selective recognition and degradation of mislocalized secretory and membrane proteins has been reconstituted in vitro. In this system, secretory and membrane proteins are synthesized in their non-translocated state simply by omitting ER-derived rough microsomes from the reaction or, if they are included, by inhibiting translocation using cotransin. With either of these manipulations, non-translocated proteins are rapidly ubiquitinated in preparation for their degradation. We are employing both classical fractionation and affinity purification approaches to identify the machinery for the selective recognition and degradation of these non-translocated proteins. In the past year, we have successfully reconstituted many of these events in vitro and have used this system to identify factors. We have identified the E2 enzyme involved, and a key chaperone that facilitates degradation. Our current aim is to identify the remaining factors of this pathway and reconstitute the events with purified components. This will pave the way for understanding mechanistically how the pathway operates. In parallel, we are moving into in vivo systems to understand the importance of this degradation pathway in maintaining cellular protein homeostasis.