Abstract
Cells employ a vast network of regulatory pathways to manage intracellular levels of reactive oxygen species (ROS). An effectual means used by cells to control these regulatory systems are sulfur-based redox switches, which consist of protein cysteine or methionine residues that become transiently oxidized when intracellular ROS levels increase. Here, we describe a methionine-based oxidation event involving the yeast cytoplasmic Hsp70 co-chaperone Fes1. We show that Fes1 undergoes reversible methionine oxidation during excessively-oxidizing cellular conditions, and we map the site of this oxidation to a cluster of three methionine residues in the Fes1 core domain. Making use of recombinant proteins and a variety of in vitro assays, we establish that oxidation inhibits Fes1 activity and, correspondingly, alters Hsp70 activity. Moreover, we demonstrate in vitro and in cells that Fes1 oxidation is reversible and is regulated by the cytoplasmic methionine sulfoxide reductase Mxr1 (MsrA) and a previously unidentified cytoplasmic pool of the reductase Mxr2 (MsrB). We speculate that inactivation of Fes1 activity during excessively-oxidizing conditions may help maintain protein-folding homeostasis in a suboptimal cellular folding environment. The characterization of Fes1 oxidation during cellular stress provides a new perspective as to how the activities of the cytoplasmic Hsp70 chaperones may be attuned by fluctuations in cellular ROS levels and provides further insight into how cells use methionine-based redox switches to sense and respond to oxidative stress.
Highlights
Cells employ a vast network of regulatory pathways to manage intracellular levels of reactive oxygen species (ROS)
We suggest that increased oxidation of a given methionine during sample preparation may indicate a methionine residue prone to modification, especially when recovery of the same oxidized methionine is further noted in peroxide-treated samples
Consistent with the mass spectrometry (MS) data, we found that the electrophoretic mobility of a Fes1 Met– to–Ile, Met–to–Phe, or Met–to–Gln triple mutant did not change when cells were exposed to cumene hydroperoxide (CHP) (Fig. 1E), supporting the conclusion that the Fes1 size shift induced by oxidant exposure is a consequence of methionine oxidation at residues Met45, Met-49, and Met-53
Summary
Cells employ a vast network of regulatory pathways to manage intracellular levels of reactive oxygen species (ROS). The characterization of Fes oxidation during cellular stress provides a new perspective as to how the activities of the cytoplasmic Hsp chaperones may be attuned by fluctuations in cellular ROS levels and provides further insight into how cells use methionine-based redox switches to sense and respond to oxidative stress. Altered protein activity can allow for the initiation of distinct downstream signaling events to benefit cells during oxidative stress, including the activation of pathways that serve to scavenge ROS before they exert detrimental effects as well as systems that act to manage and/or repair ROSincurred damage [2,3,4,5,6,7]
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