Degradation of Mislocalized Secretory and Membrane Proteins

在体外重建了对错定位分泌蛋白和膜蛋白的选择性识别和降解。在这个系统中，分泌蛋白和膜蛋白在非易位状态下合成，只需从反应中省略er来源的粗粒体，或者如果包含它们，则使用协转蛋白抑制易位。通过这两种操作，非易位蛋白迅速泛素化，为它们的降解做准备。我们采用经典的分离和亲和纯化方法来鉴定这些非易位蛋白的选择性识别和降解机制。在过去的一年里，我们已经成功地在体外重建了许多这些事件，并使用该系统来识别因素。我们已经确定了涉及的E2酶，以及促进降解的关键伴侣。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。这将为从机制上理解这一途径的运作方式铺平道路。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稳态中的重要性。

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.