Fig. 2. Met4p is destabilized specifically in repressive growth conditions. (A) Cells carrying a plasmid coding for the indicated HA–tagged proteins under the regulation of the GAL1 promoter were grown in raffinose and expression was induced by resuspending the cells in fresh galactose-containing medium (2% galactose) for 90 min. Cultures were then divided in two, filtered, transferred to glucose-containing medium in the presence or absence of 1 mM l–methionine, and samples taken at the times indicated and immunoblotted with anti-HA antibodies. The non-specific band revealed by anti-HA antibodies (indicated by an arrow in the case of Met4p stability determination) was used as a control of the amount of loaded extracts in each experiment (T = extract of cells expressing no HA-tagged protein). (B and C) Total RNA was extracted from cells expressing either the HA-Met4 or the HA-Met28 fusion proteins grown as in (A) and analyzed with MET4, MET16, MET25 and MET28 probes. (D) Induction of Met4p modification was followed by first growing the cells in raffinose-containing medium. Cells were then transferred to galactose-containing medium (2% galactose) to induce GAL1–HA-MET4 expression, and samples were taken at the times indicated.

Fig. 4. Met4p is stabilized in the presence of a cim3 mutation. (A) The stability of HA-Met4p expressed in cim3-1 mutant cells grown in repressive and non-repressive conditions was analyzed as described in Figure 3B. (B) Repression of the MET25 gene was monitored in cim3-1 and cim5-1 mutant cells. Cells were grown and RNA extracted as described in Figure 1. The wild-type (wt) strain used in this assay was the YPH499 strain, which was congenic to both mutant strains.

Fig. 6. Characterization of Met30p stability. The HA-tagged full-length Met30 and Met30ΔF proteins were expressed from a GAL1 promoter by growing the cells for 2 h in 2% galactose. Glucose (2%) and cycloheximide (50 μg/ml) were added and the cells were allowed to grow for the indicated times in the presence or absence of 1 mM l–methionine. Upper panel: the stability of both HA-Met30 and HA–Met30ΔF was monitored in W303-1A wild-type cells. Lower panel: the stability of HA-Met30 was monitored in the SCF mutants skp1-11 and cdc53-1 by shifting the cells to 37°C before the addition of galactose as well as in met4Δ mutant cells.

Fig. 8. Relationships between the trans-acting factors regulating the MET network and how AdoMet-induced degradation of the Met4p activator is triggered by the SCF^Met30 complex.

Fig. 1. Regulation of the sulfate assimilation pathway in SCF^Met30 mutants. The strains were grown for eight generations in B medium with 0.2 mM dl-homocysteine as sulfur source, shifted to 37°C for 2 h and a repressing amount of l-methionine (1 mM) was then added. Total RNA was extracted at the indicated times after l-methionine addition, and expression of MET genes was determined by Northern blot analysis. The actin probe was used as a control of the amount of RNA loaded.

Fig. 3. Met4p degradation depends on the SCF^Met30 complex. (A) The stability of HA-Met4Δinh expressed in wild-type cells grown in repressive and non-repressive conditions was analyzed as described in Figure 2A. (B) The stability of HA-Met4p was studied in SCF^Met30 mutants: wild-type and mutant cells were grown in raffinose at 28°C for eight generations, filtered and resuspended in galactose medium for 30 min at 28°C. Cells were then shifted to 37°C and incubation in galactose medium was continued for another 60 min. Cells were then transferred to fresh glucose medium containing 1 mM l-methionine and proteins were extracted at the indicated times and processed for immunoblotting with anti-HA antibodies. Equal loading was determined by the presence of the non-specific band revealed by the anti-HA antibodies (not shown). (C and D) GFP–Met4 localization was monitored in wild-type cells and in SCF^Met30 mutants. Cells expressing the GFP–Met4 fusion protein from the GAL1 promoter were grown in raffinose at 28°C, filtered and resuspended in galactose medium for 60 min at 28°C. Cells were then shifted to 37°C and incubation in galactose medium was continued for another 60 min. In repressive growth conditions (+Met), 1 mM l-methionine was added 15 min before observation. Nuclei were visualized by adding the dye Hoescht 33342 (Hoe) to the culture 20 min before observation (Nom = Nomarski interferential observation). (E) Localization of the GFP–Met28 derivative was monitored in cells expressing the fusion protein from the MET28 promoter and grown in the presence (+Met) or absence (–Met) of a repressing amount of l-methionine (1 mM).

Fig. 5. Met4p–Met30p interactions. (A) Co-immunoprecipitation analysis of the Met4p–Met30p interaction. HA-Met4p and GST–Met30p were expressed, alone or together, from the GAL1 promoter by growing the cells for 2 h in the presence of 2% galactose. In non-repressive growth conditions, anti-HA immunoprecipitates were probed with either anti-HA or anti-GST antibodies, while anti-GST antibodies revealed the presence of the GST–Met30 fusion protein in the lysates. (B) Two-hybrid analysis of the interaction established between Gal4–Met4 and LexA–Met30 fusion proteins. β-galactosidase activities were revealed on X-gal plates containing ammonium sulfate as the sulfur source. (C) Western assays of LexA–Met30 derivatives. Proteins were extracted from cells expressing the various LexA–Met30 derivatives and processed for immunoblotting with anti-LexA antibodies. The asterisk indicates the non-specific band revealed by the anti-LexA antibodies. (D) Met4p is not modified by incubation in the presence of alkaline phosphatase. Immunoprecipitates of HA-Met4 were incubated in the presence of 20 or 40 U of alkaline phosphatase (CIP) at 37°C for 15 min. Incubations were loaded onto a gel and probed with anti-HA antibodies. As a control, an immunoprecipitate of an HA-Cln2 fusion protein expressed from the GAL1 promoter was incubated in the presence of 20 U of CIP at 37°C for 15 min. (E) GFP–Met30 localization. The GFP–Met30 fusion protein was expressed from the GAL1 promoter and its localization in living cells was monitored as in Figure 3C in the presence or absence of 1 mM l-methionine. (F) Skp1p–Met30p interaction is not affected by methionine concentration. Cells expressing HA-Met30 protein from the ADH1 promoter were grown in rich (XY), non-repressive (NR) or repressive (R) media conditions. Western blots of lysates and anti-HA immunoprecipitates were probed with anti-HA antibodies and anti-Skp1 antibodies. ‘+’ and ‘–’ denote the presence or absence of the protein or antibody.

Fig. 7. MET30 transcription is regulated by intracellular AdoMet and Met4p. (A) Derepression kinetics of MET3, MET4, MET16 and MET30 transcription were monitored in wild-type and met4Δ cells. Cells were grown in 100 ml of B medium in the presence of a repressing amount (1 mM) of l-methionine as sulfur source. When the cells reached a density of ∼10⁷ cells/ml, they were harvested by filtration and washed with 100 ml of B medium. The cells were then suspended in 100 ml of B medium without methionine and shaken at 28°C. Total RNA was extracted at the times indicated. The actin probe was used as a control of the amounts of RNA loaded. (B) Gel retardation assays were performed with a MET30 radiolabeled probe corresponding to nucleotides –255 to –61. The binding reactions were performed with 40 μg extracts from strains W303-1A (wild–type), CD106 (met4::TRP1), CD130-7D (met28::LEU2) and CD179 (met31::LEU2, met32::TRP1). (C) The binding reactions were performed with 40 μg extracts from strains W303-1A (wild-type) and 60 μg extracts from strains CC857-2A (cbf1::TRP1) and CC653-3C (met4::TRP1, cbf1::TRP1). (D) The binding reactions were performed with extracts from strains W303-1A (wild-type), CD106 (met4::TRP1) and CD179 (met31::LEU2, met32::TRP1), and from strains CD106 and CD179 expressing, respectively, a LexA–Met4 or a LexA–Met32 fusion protein. All the reactions were carried out in the presence of 40 μg of proteins. Where indicated, 1 μl of a polyclonal antibody raised against LexA used at a 1/10 dilution was added. Arrows indicate the Met4-containing high molecular weight complexes. (E) The stability of HA-Met4 was assayed in the met31Δ, met32Δ mutant cells grown in the absence (–Met) or presence (+ Met) of 1 mM l-methionine. Experiments were carried out as described in Figure 2A. (F) Induction of Met4p in met31Δ, met32Δ mutant cells was followed by first growing the cells in raffinose-containing medium. Cells were then transferred to galactose-containing medium (2% galactose) to induce GAL1–HA-MET4 expression and samples were taken at the times indicated.