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

点击翻译按钮获取中文摘要

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.