Characterization of the Molecular Chaperone Get3

by Wilhelm Voth
Doctoral thesis
Date of Examination:2016-09-20
Date of issue:2017-08-03
Advisor:Prof. Dr. Blanche Schwappach
Referee:Prof. Dr. Blanche Schwappach
Referee:Prof. Dr. Jakob Ursula
Persistent Address: http://dx.doi.org/10.53846/goediss-6410

 

 

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Abstract

English

Proteins within the cellular environment are constantly challenged by environmental stress conditions that threaten their structure and function, and increase the risk of forming potentially toxic protein aggregates. Sudden exposure of cells to increased levels of reactive oxygen species (ROS) beyond the limits of the cellular detoxification machinery causes a rapid and significant drop in intracellular ATP-levels disturbing the cellular proteostasis network. This mostly ATP-dependent chaperone system becomes inactive under these conditions, making ROS-mediated protein unfolding and aggregation a potentially very challenging problem for organisms. Here we demonstrate that the yeast protein Get3, involved in ATP-dependent targeting of tail-anchored (TA) proteins under non-stress conditions, turns into an effective ATP-independent general chaperone under oxidative stress conditions. The process of Get3’s activation as chaperone is reminiscent of Hsp33, the prokaryotic paradigm for a redox-regulated chaperone. It involves disulfide bond formation, zinc release and the conversion of Get3 into distinct, higher oligomeric structures which adopt an ATP-independent chaperone holdase function. Most importantly, the substantial structural rearrangements that expose binding sides for unfolded protein upon oxidation are fully reversible. Restoration of non-stress conditions return Get3 into an ATP-binding targeting factor for tail-anchored proteins. Unfolded substrates bound to chaperone active Get3 are then released and transferred to the ATP-dependent folding machinery for their refolding. Mutational studies revealed that Get3’s chaperone activity is functionally distinct from and likely mutually exclusive with its targeting function. Indeed, in vivo complementation studies demonstrated that the oxidative stress sensitive phenotype that has long been observed for yeast cells lacking functional Get3 is specifically due to the absence of Get3’s chaperone function. Our results provide convincing evidence that Get3 moonlights as a redox regulated chaperone in the proteostasis network of eukaryotic cells, effectively protecting proteins against oxidative damage and allowing their refolding upon return to non-stress conditions.
Keywords: Redox regulation; GET pathway; Molecular Chaperones; Oxidative stress
 
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