Evidence for a hydrogen sulfide-sensing E3 ligase in yeast

Abstract

In yeast, control of sulfur amino acid metabolism relies upon Met4, a transcription factor that 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 trans-sulfuration 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 trans-sulfuration pathway to regulate the synthesis of these special amino acids.

Keywords: E3 ubiquitin ligase; amino acid; metabolism; nutrients; sensor; sulfur; yeast.

Fig. 1.

Met30 and Met4 response to sulfur starvation and repletion under respiratory growth conditions. a) Schematic of experimental regimen used throughout this study. All sulfur sources were depleted for the indicated times, followed by supplementation of the designated sulfur sources to the same culture for the indicated times. b) Western blot analysis of Met30 and Met4 over the sulfur starvation time course. Yeast containing endogenously tagged Met30 and Met4 were cultured in rich lactate media (Rich) overnight to mid-log phase before switching cells to sulfur-free lactate media (−sulfur) for 1 h, followed by the addition of a mix of the sulfur-containing metabolites methionine, homocysteine, and cysteine at 0.5 mM each (+Met/Cys/Hcy). Rpn10 is used as the loading control. The blot shown is representative of 3 replicate experiments. c) Expression of MET gene transcript levels was assessed by qPCR over the time course shown in (a). Data are presented as mean and SEM of technical triplicates. d) Levels of key sulfur metabolites were measured over the same time course as in (a) and (b), as determined by LC-MS/MS. Data represent the mean and SD of 2 biological replicates.

Fig. 2.

Synthesis of cysteine is more important than methionine for Met4 ubiquitination. a) Simplified diagram of the yeast sulfur amino acid biosynthetic pathway. b) Western blots of Met4 ubiquitination status in response to rescue with various sulfur metabolites in the methyl cycle in WT, sah1Δ, and sam1Δ/sam2Δ strains. Cells were grown in “Rich” YPL and switched to “−sulfur” SFL for 1 h to induce sulfur starvation before the addition of either 0.5 mM homocysteine (+Hcy), 0.5 mM methionine (+Met), or 0.5 mM S-adenosylmethionine (+SAM). c) Western blots of Met4 ubiquitination status in met6Δ or str3Δ strains in response to rescue with various sulfur metabolites. Cells were grown in “Rich” YPL and switched to “−sulfur” SFL for 1 h to induce sulfur starvation before the addition of either 0.5 mM homocysteine (+Hcy), 0.5 mM methionine (+Met), or 0.5 mM cysteine (+Cys). d) Western blots of Met4 ubiquitination status in WT, cys3Δ and cys4Δ strains produced in the S288C background in response to rescue with sulfur metabolites in the trans-sulfuration pathway. Cells were grown in sulfur-free glucose media supplemented with methionine to log phase before removing methionine and switching to sulfur-free glucose media for 3 h to induce sulfur starvation. Subsequently, either 0.5 mM homocysteine (+Hcy) or 0.5 mM cysteine (+Cys) was added to cells.

Fig. 3.

Hydrogen sulfide is a signal for sulfur sufficiency. a) Western blot analysis of Met30 and Met4 ubiquitination in WT and met10Δ/met17Δ yeast grown in “Rich” YPL and switched to “−sulfur” SFL for 1 h to induce sulfur starvation before the addition of 20 µM disodium sulfide (Na₂S). Note that the met10Δ/met17Δ strain cannot produce sulfide from inorganic sulfate or utilize exogenous sulfide to produce homocysteine. b) Levels of key sulfur metabolites were measured over the same time course as in (a), as determined by LC-MS/MS. Data represent the mean and SD of two biological replicates.

Fig. 4.

Met30 cysteine point mutants display dysregulated sulfur sensing. a) Schematic of Met30 protein architecture and cysteine residue location. b) Western blot analysis of Met30 and Met4 ubiquitination status in WT and two cysteine to serine mutants, C414S and C614/616/622/630S. c) MET gene transcript levels over the same time course as (a) for the three strains, as assessed by qPCR. Data are presented as mean and SEM of biological triplicates. d) Growth curves of the three yeast strains used in (a) and (b) in sulfur-rich YPL media or −sulfur SFL media supplemented with 0.2 mM homocysteine. Cells were grown to mid-log phase in YPL media before pelleting, washing with water, and back-diluting yeast into the two media conditions. Data represent mean and SD of biological triplicates.

Fig. 5.

Model for sulfur-sensing and MET gene regulation by the SCF^Met30 E3 ligase. In conditions of high sulfur-containing amino acids and related metabolites and possibly sulfide levels, cysteine residues in the WD-40 repeat region of Met30 are reduced or potentially persulfidated, allowing Met30 to bind and facilitate ubiquitination of Met4 in order to inhibit the transcriptional activation of the MET regulon. Upon sulfur starvation, Met30 releases Met4 to be deubiquitinated and activate the MET gene transcriptional program.