A conserved and multifunctional redox switch in yeast Get3 and mammalian TRC40

在氧化应激期间维持蛋白质的动态平衡是所有生物体面临的主要挑战。这是由于活性氧大大降低了细胞的ATP水平，从而使现有的依赖于ATP的伴侣丧失能力，并减缓了其他依赖于ATP的过程。我们之前的研究有助于认识到，生物体使用一类非常不同的应激特异的ATP非依赖性伴侣蛋白，这些伴侣蛋白在翻译后被氧化应激激活。我们发现，Get3(哺乳动物中的TRC40)是尾部锚定蛋白(TA-Protein)插入机制中的一个可溶性成员，受硫醇开关的氧化还原调节，并在氧化应激介导的二硫键形成过程中迅速转变为有效的分子伴侣。依赖于ATP的伴侣功能的激活伴随着依赖于ATP的TA蛋白靶向活性的丧失，使Get3成为真正的双功能蛋白。在酵母中使用功能性Get3变体进行的互补研究表明，许多先前观察到的get3缺失表型是由Get3的伴侣活性而不是它的TA插入功能造成的。这些结果特别令人兴奋，因为它们暗示Get3的伴侣功能实际上可能是Get3在生理上更重要的角色。我们现在将应用我们在细菌氧化还原调节的伴侣蛋白Hsp33上开发的广泛的机制和结构工具箱来研究Get3/TRC40在体外和体内的氧化还原调节的激活。我们将特别关注可溶性和膜基GET复合体的其他成员在Get3伴侣功能中的作用，假设这些蛋白质促进Get3的功能变化，影响客户结合和/或释放。我们将在酵母中验证我们的体外结果，特别是利用我们之前产生的许多受到严格操纵和特性良好的酵母突变体，并在哺乳动物细胞系中测试相应的突变体，使用已建立的条件，通过siRNA介导的TRC40机械的各种组件的击倒。我们将在酵母中使用不同生理条件下的系统定位筛选来识别完全依赖Get3伴侣功能的客户蛋白，并将监测这些蛋白在应激和应激恢复期间的命运。这些结果将为Get3/TRC40在酵母和哺乳动物中扮演的促进生存的角色提供重要的新见解。

Maintaining protein homeostasis during oxidative stress is a major challenge for all organisms. This is due to the fact that reactive oxygen species substantially decrease cellular ATP levels, therefore incapacitating existing ATP-dependent chaperones and slowing down other ATP-dependent processes. Our previous studies have contributed to the realization that organisms use a very different class of stress-specific ATP-independent chaperones, which are posttranslationally activated by oxidative stress. We discovered that Get3 (TRC40 in mammals), a soluble member of the tail-anchored protein (TA-protein) insertion machinery, is redox-regulated by a thiol switch, and rapidly turns into an effective molecular chaperone upon oxidative stress-mediated disulfide bond formation. Activation of the ATP-independent chaperone function goes hand in hand with a loss in ATP-dependent TA-protein targeting activity, making Get3 a bona fide dual-function protein. Complementation studies in yeast using functional Get3 variants revealed that the chaperone activity of Get3 and not its TA-insertion function is responsible for many of the previously observed get3 deletion phenotypes. These results are particularly exciting since they imply that the chaperone function of Get3 might in fact be the physiologically more significant role of Get3. We will now apply the extensive mechanistic and structural toolset that we have developed in our work on the bacterial redox-regulated chaperone Hsp33 to investigate the redox-regulated activation of Get3/TRC40 both in vitro and in vivo. We will pay particular attention to the role of the other members of the soluble and membrane-based GET complexes in the chaperone function of Get3, working on the assumption that these proteins promote functional changes in Get3, affect client binding and/or release. We will validate our in vitro results in yeast, making particular use of the many heavily manipulated and well-characterized yeast mutants that we have previously generated, and test corresponding mutants in mammalian cell lines using established conditions for the siRNA-mediated knock-down of various components of the TRC40 machinery. We will use a systematic localization screen in yeast under different physiological conditions to identify client proteins that rely exclusively on the chaperone function of Get3, and will monitor the fate of these proteins during stress and upon stress recovery. These results will provide important new insights into the pro-survival role that Get3/TRC40 plays in yeast and mammals.