Activity of the yeast cytoplasmic Hsp70 nucleotide-exchange factor Fes1 is regulated by reversible methionine oxidation.

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.

The forkhead transcription factor UNC-130/FOXD integrates both BMP and Notch signaling to regulate dorsoventral patterning of the C. elegans postembryonic mesoderm.

The proper development of a multicellular organism requires precise spatial and temporal coordination of cell intrinsic and cell extrinsic regulatory mechanisms. Both Notch signaling and bone morphogenetic protein (BMP) signaling function to regulate the proper development of the C. elegans postembryonic mesoderm. We have identified the C. elegans FOXD transcription factor UNC-130 as a major target functioning downstream of both BMP signaling and Notch signaling to regulate dorsoventral patterning of the postembryonic mesoderm. We showed that unc-130 expression in the postembryonic M lineage is asymmetric: its absence of expression in the dorsal side of the M lineage requires the antagonism of BMP signaling by the zinc finger transcription factor SMA-9, while its expression in the ventral side of the M lineage is activated by LIN-12/Notch signaling. We further showed that the regulation of UNC-130 expression by BMP signaling and Notch signaling is specific to the M lineage, as the ventral expression of UNC-130 in the embryonically-derived bodywall muscles was not affected in either BMP pathway or Notch pathway mutants. Finally, we showed that the function of UNC-130 in the M lineage is independent of UNC-129, a gene previously shown to function downstream of and be repressed by UNC-130 for axon guidance. Our studies uncovered a new function of UNC-130/FOXD in the C. elegans postembryonic mesoderm, and identify UNC-130 as a critical factor that integrates two independent spatial cues for the proper patterning and fate specification of the C. elegans postembryonic mesoderm.