ABSTRACT
In yeast, control of sulfur amino acid metabolism relies upon Met4, a transcription factor which activates the expression of a network of enzymes responsible for the biosynthesis of cysteine and methionine. In times of sulfur abundance, the activity of Met4 is repressed via ubiquitination by the SCFMet30 E3 ubiquitin ligase, but the mechanism by which the F-box protein Met30 senses sulfur status to tune its E3 ligase activity remains unresolved. Herein, we show that Met30 responds to flux through the transsulfuration pathway to regulate the MET gene transcriptional program. In particular, Met30 is responsive to the biological gas hydrogen sulfide, which is sufficient to induce ubiquitination of Met4 in vivo. Additionally, we identify important cysteine residues in Met30’s WD-40 repeat region that sense the availability of sulfur in the cell. Our findings reveal how SCFMet30 dynamically senses the flow of sulfur metabolites through the transsulfuration pathway to regulate synthesis of these special amino acids.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
During the COVID-19 pandemic, we have found that different TCAs from different vendors, as well as TCAs between catalogues and lots from the same vendor, can result in unusual and variable cysteine labeling by thiol-modifying reagents. Previous data we have generated suggested profound redox changes in Met30 cysteines over the sulfur starvation time course that are not detectable when using different batches of trichloroacetic acid. While we have tried, major chemical suppliers have been reluctant to disclose the suppliers for the different catalogue numbers and lots of TCA that we have experimented with, so we have been unable to trace the problem to a particular supplier. Therefore, caution should be asserted with respect to thiol labeling following trichloroacetic acid quenching of cells and proteins. More research is needed to understand the source of variability with different lots of TCA, as well as to develop alternative methods to preserve and label cysteines in such a way as to reliably preserve their in vivo redox state. Therefore, we have withdrawn the TCA experiments that were no longer reproducible, which led to alternate interpretations of certain data, and we have updated our findings accordingly in this revision.