Substrate-mediated remodeling of methionine transport by multiple ubiquitin-dependent mechanisms in yeast cells

Abstract

Plasma membrane transport of single amino-acid methionine in yeast is shown to be mediated by at least seven different permeases whose activities are transcriptionaly and post-transcriptionaly regulated by different ubiquitin-dependent mechanisms. Upon high extracellular methionine exposure, three methionine-permease genes are repressed while four others are induced. SCF(Met30), SCF(Grr1) and Rsp5 ubiquitin ligases are the key actors of the ubiquitin-dependent remodeling of methionine transport. In addition to regulating the activity of Met4, the SCF(Met30) ubiquitin ligase is shown to convey an intracellular signal to a membrane initiated signaling pathway by controlling the nuclear concentration of the Stp1 transcription factor. By coupling intra- and extracellular metabolite sensing, SCF(Met30) thus allows yeast cells to accurately adjust the intermediary sulfur metabolism to the growth conditions. The multiple ubiquitin-dependent mechanisms that function in methionine transport regulation further exemplify the pervasive role of ubiquitin in the adaptation of single-cell organisms to environmental modifications.

Figure 1

Met4 and the SPS sensor control methionine permease gene expression. Methionine permease gene expression was assessed by quantitative real-time RT–PCR in (A) wild-type (CD269), (B) met4Δ (CC849-8A) and (C) ssy1Δ (CD317) cells grown in the absence and presence of 1 mM L-methionine. Values represent the average of two independent experiments and error bars indicate standard deviations. (D) Wild-type (W303-1A), ssy5Δ (CD319), met3Δ (CY14-5D), met4Δ (CC849-8A), ssy5Δ met3Δ (CY343-1C) and ssy5Δ met4Δ (CY33-2B) cells were plated on minimal medium supplemented with 0.1 mM L-methionine or 0.5 mM AdoMet.

Figure 2

Methionine permeases are controlled by the Stp1, Stp2 and Met4 transcription activators. Methionine permease gene expression was assessed in (A) wild-type (CD269), (B) stp1Δ stp2Δ (CD354) and (C) in uga35Δ (CD357) as in Figure 1. (D) Serial dilutions of wild-type (W303-1A), met4Δ (CC849-8A), stp1Δ stp2Δ (CD354), stp1Δ stp2Δ met4Δ (CY350-1C), uga35Δ (CD357) and met4Δ uga35Δ (CY353-16C) cells were plated on minimal medium in the absence and presence of either 0.1 mM L-methionine or 0.2 mM AdoMet.

Figure 3

Met4, Stp1 and TFIIB occupancy at methionine permease promoters. CD362 (SSY1, stp1∷STP1^HA, sua7∷SUA7^MYC9) and CD364 (ssy1Δ, stp1∷STP1^HA, sua7∷SUA7^MYC9) cells were grown in minimal B medium and exposed to 1 mM L-methionine. After crosslinking, the chromatin was immunoprecipitated with either anti-Met4 (A), anti-HA (B) or anti-Myc (C) antibody. Total DNA was analyzed by quantitative PCR with primer-pairs-specific promoters. IME2 ORF was used as a control. The indicated IP/total ratio corresponds to the concentration of target DNA in the immunoprecipitated sample relative to that in the corresponding input sample. The reported values represent the average of two independent experiments, and error bars indicate standard deviations.

Figure 4

Regulation of the methionine permease genes depends on both SCF^Met30 and SCF^Grr1 ubiquitin ligases. (A) Met4-dependent methionine permease gene expression was analyzed by quantitative RT–PCR in met30-10 (CY213-8A) and grr1Δ (CD344) cells exposed or not to 1 mM L-methionine. Total RNA was extracted before and 40 min after methionine addition. MET25 gene expression was used as a control of methionine repression. (B) SPS-dependent methionine permease gene expression was analyzed by quantitative RT–PCR in wild-type (W303-1A), met30-10 (CY213-8A) and grr1Δ (CD344) cells exposed or not to high extracellular methionine (1 mM). Total RNA was extracted before and 40 min after methionine addition. (C) Met30 regulates the binding of Stp1 on the AGP1, BAP2, BAP3 and GNP1 promoters. CD384 (stp1∷STP1^HA) and CD383 (met30-10, stp1∷STP1^HA) cells were grown in minimal B medium and exposed to 1 mM L-methionine for 40 min. ChIPs were performed as in Figure 3. (D) Addition of Cd²⁺ to the medium compromised the methionine-mediated regulation of both classes of methionine permeases. Total RNA was extracted before and 40 min after the addition of 1 mM L-methionine, in the absence and presence (+Cd²⁺) of 100 μM Cd²⁺, and analyzed by quantitative RT–PCR.

Figure 5

High methionine targets Stp1 to the nucleus. (A) CD362 (SSY1, stp1∷STP1^HA) and CD364 (ssy1Δ, stp1∷STP1^HA) cells were grown in minimal B medium and exposed to 1 mM L-methionine. Total proteins were extracted and processed for immunoblotting with monoclonal antibody to Ha and polyclonal antibody to the yeast lysyl-tRNA synthetase. (B) Methionine induces Stp1 nuclear localization. The Stp1-GFP^Venus fusion protein was expressed in the SSY1 (CD386) and ssy1Δ (CD387) cells from the GAL1 promoter for 1 h. To limit the expression level of the Stp1-GFP^Venus fusion protein, the cells were transferred to a glucose-containing medium to repress the GAL1 promoter. The culture was then split into two cultures, and the cells grown for 40 min, either in the absence of methionine (−) or in the presence of 1 mM L-methionine (+Met). Fluorescence images were then acquired. The fluorescence dye Hoesch labels the nucleus. (C) Stp1 is localized in the nucleus upon methionine exposure. Localization of the tagged Stp1^3HA protein expressed from the endogenous promoter was analyzed by immunofluorescence in CY352 cells exposed or not to 1 mM L-methionine for 40 min. (D) Constitutive nuclear localization of Stp1 in met30Δ met32Δ mutant cells. The Stp1-GFP^Venus protein was expressed in the met30Δ met32Δ mutant cells (CD396) from the GAL1 promoter. Cells were grown as in (B). Images were acquired in the absence of methionine (−) and in the presence of 1 mM methionine (+Met). (E) Cd²⁺ exposure leads to Stp1 nuclear accumulation. Localization of the tagged Stp1^3HA protein was analyzed by immunofluorescence in CD362 cells exposed or not to Cd²⁺.

Figure 6

Stp1 nuclear targeting depends upon an intracellular sulfur-containing compound. (A) Methionine permease gene expression was analyzed by quantitative real time RT–PCR in wild-type cells (CD269) grown in minimal medium before and after addition of either 0.2 or 0.5 mM AdoMet. (B) CD362 (SSY1, stp1∷STP1^HA) and CD364 (ssy1Δ, stp1∷STP1^HA) cells were grown in minimal B medium and exposed to 0.5 mM AdoMet. Total proteins were extracted and processed as in Figure 5. (C) High extracellular AdoMet induces Stp1 nuclear localization. The Stp1-GFP^Venus fusion protein was expressed as in Figure 5B except that 0.5 mM AdoMet, instead of 1 mM methionine, was added into the medium. (D) SPS-dependent methionine permease genes expression was assessed by quantitative real-time RT–PCR analysis in sam3Δ cells (CD192) exposed or not to either1 mM methionine or 0.5 mM AdoMet. (E) High intracellular AdoMet induces Stp1 nuclear accumulation. The Stp1-GFP^Venus fusion protein was expressed in sam3Δ (CY356-3B) and sam1Δ sam2Δ (CD398) cells exposed to either 1 mM methionine or 0.5 mM AdoMet. (F) Intracellular conversion of AdoMet into cysteine is not required for Stp1 nuclear accumulation. The Stp1-GFP^Venus fusion protein was expressed in str4Δ (CD400) cells exposed to either 1 mM methionine (+Met), 1 mM cysteine (+Cys) or 0.5 mM AdoMet (+AdoMet).

Figure 7

High extracellular methionine induces the ubiquitin-dependent destabilization of Mup1-Gfp fusion protein. (A) Destabilization of the Mup1-Gfp fusion protein in living cells exposed to high methionine levels. CD378 (mup1∷MUP1-GFP) cells were grown in the absence (−Met) or in the presence of 1 mM L-methionine (+Met). Cells were imaged at the indicated times after methionine addition. (B) Stabilization of the Mup1-Gfp fusion protein in a rsp5 mutant. CD379 (mup1∷MUP1-GFP, rsp5-1) cells were grown in minimal B medium, shifted to the nonpermissive temperature 37°C for 2 h and then exposed or not to 1 mM methionine. Cells were imaged at the indicated times after the addition of methionine (+Met) or not (−Met). (C) Stabilization of the Mup1-Gfp fusion protein in a doa4 mutant. CD380 (mup1∷MUP1-GFP, doa4∷LEU2) cells were grown in the absence (−Met) or in the presence of 1 mM methionine (+Met).