Inducible dissociation of SCF(Met30) ubiquitin ligase mediates a rapid transcriptional response to cadmium

Abstract

Activity of the Met4 transcription factor is antagonized by the SCF(Met30) ubiquitin ligase by degradation-dependent and degradation-independent mechanisms, in minimal and rich nutrient conditions, respectively. In this study, we show that the heavy metal Cd2+ over-rides both mechanisms to enable rapid Met4-dependent induction of metabolic networks needed for production of the antioxidant and Cd2+-chelating agent glutathione. Cd2+ inhibits SCF(Met30) activity through rapid dissociation of the F-box protein Met30 from the holocomplex. In minimal medium, dissociation of SCF(Met30) complex is sufficient to impair the methionine-induced degradation of Met4. In rich medium, dissociation of the SCF(Met30) complex is accompanied by a deubiquitylation mechanism that rapidly removes inhibitory ubiquitin moieties from Met4. Post-translational control of SCF(Met30) assembly by a physiological stress to allow rapid induction of a protective gene expression program represents a novel mode of regulation in the ubiquitin system.

Figure 1

Cd²⁺inhibits the degradation-dependent regulation of Met4. (A) Cd²⁺ prevents MET gene repression by methionine in minimal medium. Total RNA was extracted at the indicated times after the addition of 1 mM L-methionine, in the presence (+Cd²⁺) or absence of 100 μM Cd²⁺, or in the presence of 100 μM of both Cd²⁺ and EGTA (+Cd²⁺, +EGTA), and MET gene expression was assessed by Northern analysis. (B) Cd²⁺ blocks elimination of endogenous GFP-Met4 by methionine. met4∷GFP-MET4 cells (strain CD240) were imaged at the indicated times after the addition (+Met) or not (−Met) of 1 mM L-methionine, in the presence (+Cd²⁺) or absence of 100 μM Cd²⁺, or in the presence of 100 μM of both Cd²⁺ and EGTA (+Cd²⁺, +EGTA). (C) Cd²⁺ stabilizes endogenous Met4 protein. met4∷^HA3MET4 cells (strain CD233) were treated with (+Met) 1 mM L-methionine, in the presence (+Cd²⁺) or absence of 100 μM Cd²⁺, and total protein from TCA extracts was immunoblotted for the HA epitope or, as a control, lysyl-tRNA synthetase. (D) Met4p and TFIIB occupancy at MET promoters is elevated in response to Cd²⁺ exposure. A strain expressing both ^HA3Met4 and TFIIB^9MYC (CD269) or an untagged isogenic control strain (W303-1A) were grown in B minimal medium and exposed to 1 mM L-methionine, in the presence or absence of 100 μM Cd²⁺. After crosslinking and immunoprecipitation with anti-HA or anti-Myc antibody, total DNA was analyzed by quantitative PCR with primer-pairs specific to the indicated Pol II promoters. ChIPs were performed at least twice and yielded similar results.

Figure 2

Cd²⁺ activates MET gene expression in rich medium. (A) Rapid induction of MET genes by Cd²⁺. MET gene expression in wild-type cells was assessed at the indicated times after the addition of 0.5 mM Cd²⁺ in the absence or presence of 0.5 mM EGTA. (B) Cd²⁺-mediated activation of MET genes depends on Met4. MET gene expression was assessed in wild-type and met4Δ (CC849-8A) cells at the indicated times after the addition of 0.5 mM Cd²⁺. (C) Met4 and TFIIB recruitment to MET gene promoters in response to Cd²⁺. ChIP experiments were carried out as in Figure 1 on wild-type cultures before and 40 min after the addition of 0.5 mM Cd²⁺. (D) Cd²⁺ specifically induces MET gene expression. MET gene expression was assessed in wild-type cells before and 40 min after addition of the indicated heavy metals by Northern analysis (left) and quantitative real-time RT–PCR (right). Heavy metals were added at the following concentrations: Cu²⁺ 1 mM, Zn²⁺ 1 mM, Co²⁺ 2 mM, Hg²⁺ 0.3 μM, Mn²⁺ 0.5 mM and Ag²⁺ 30 μM.

Figure 3

Cd²⁺ impairs SCF^Met30-dependent ubiquitylation of Met4 in rich medium. (A) Collapse of Met4 isoforms in response to Cd²⁺. met4∷^HA3MET4 cells (strain CD233) were exposed to 0.5 mM Cd²⁺ in the absence or presence of 0.5 mM EGTA and extracted proteins analyzed for Met4 and lysyl-tRNA synthetase abundance by immunoblot. (B) Loss of ubiquitinated Met4 species upon Cd²⁺ treatment. Endogenous Met4 was immunoprecipitated with anti-Met4 antibody and then immunoblotted with anti-Met4 and anti-ubiquitin antibodies. (C) Accumulation of phosphorylated Met4 species in response to Cd²⁺ and in the absence of Met30. Wild-type, met4Δ and met4∷GAL1-MET4, met30Δ strains were grown in rich medium containing 2% raffinose and harvested before and after 40 min of the indicated treatment with either 0.5 mM Cd²⁺ or galactose. Anti-Met4 immunoprecipitates were treated with lambda phosphatase in the presence or absence of phosphatase inhibitors and then immunoblotted with anti-Met4 antibody.

Figure 4

Cd²⁺ induces dissociation of the SCF^Met30 ubiquitin ligase. (A) Met30 half-life is not altered by Cd²⁺ exposure. A strain bearing a GAL1-^HA3MET30 construct was induced in galactose medium for 2 h, then transferred from galactose to 2% glucose- and 1 mM methionine-containing medium, in the presence or absence of 100 μM Cd²⁺, and ^HA3Met30 abundance assessed by anti-HA immunoblot at the indicated times after glucose repression. (B) The Met4–Met30 interaction is unaffected by Cd²⁺. An ^HA3Met30 fusion protein was expressed from a GAL1 promoter in B medium containing 2% galactose in the presence or absence of 100 μM Cd²⁺, Met4 immunoprecipitated from extracts with anti-Met4 antibody and immunoblotted with anti-Met4 and anti-HA antibodies. (C) The Skp1–Met30 interaction is specifically abolished by Cd²⁺ in minimal medium. ^HA3Met30 and ^HA3Cdc53 fusion proteins were expressed as in (B), extracts subjected to immunoprecipitation with the indicated antibodies and immunoblotted with anti-Skp1 and anti-HA antibodies. (D) The Skp1–Met30 interaction is specifically abolished upon Cd²⁺ exposure in rich medium. The experiments were performed as in (C) but cells were grown in rich medium and in the presence of 0.5 mM Cd²⁺. (E) The Skp1–Cdc4 interaction is not abolished upon Cd²⁺ exposure in rich medium. Met30^MYC3 and Cdc4^MYC3 fusion proteins were expressed from the constitutive CDC53 promoter (Ho et al, 2002) in rich medium in the presence or absence of 0.5 mM Cd²⁺ and protein interactions tested as in (C) with anti-MYC and anti-Skp1 antibodies. (F) Sic1 instability is not affected by Cd²⁺. A strain (CYS37) expressing a GFP-Sic1 fusion protein from the GAL1 promoter was grown in minimal medium, arrested in G1 phase by α-factor mating pheromone for 1.5 h, released into glucose medium in the presence and absence of 100 μM Cd²⁺, with or without α-factor, and GFP-Sic1 abundance assessed 120 min after the promoter shutoff by immunoblot with anti-GFP or, as a control, anti-lysyl-tRNA synthetase (LysRS) antibodies. (G) Quantification by flow cytometry of GFP-Sic stability in either an α-factor block or after release from the block in the presence and absence of 100 μM Cd²⁺. Cells were grown as described in (F) and the GFP-Sic1 fluorescent signal recorded and quantified at the indicated times using a Beckton Dickinson FacScalibur™ flow cytometer. (H) Met4 ubiquitylation by recombinant SCF^Met30 is not affected by Cd²⁺. Purified recombinant Met4 and SCF^Met30 produced in insect cells were incubated with E1 enzyme, Cdc34, ubiquitin and ATP, either in the absence or presence of the indicated amounts of Cd²⁺. Methyl-ubiquitin served as a control to demonstrate polyubiquitin chain formation on Met4.

Figure 5

Cd²⁺ triggers deubiquitylation of Met4 in rich medium in the absence of de novo Met4 synthesis. (A) Schematic representation of the experiment. (B) Cd²⁺-activated MET gene expression does not require de novo Met4 synthesis. A met4∷GAL1-MET4 strain (CC932-6D) was subjected to the regimen in (A) and assessed for MET gene expression at the indicated time points. (C) Cd²⁺ induces Met4 deubiquitylation. The same experiment as in (B) was performed except that Met4 was immunoprecipitated and immunoblotted with anti-Met4 antibody. (D) Inactivation of SCF^Met30 does not induce Met4 deubiquitylation. Cultures of wild-type and cdc53-1 strains were shifted to the nonpermissive temperature of 37°C at the same time as repression of GAL1-MET4 by glucose. Cd²⁺ was either added or not at 60 min postshift.

Figure 6

Cd²⁺ sensitivity of met4Δ and met31Δ met32Δ strains. Serial dilutions of wild-type (WT, W303-1A), met28Δ (CC769-7D), met4Δ (CC849-1B), met31Δ (CC867-1C), met32Δ (CC845-1C) and met31Δ met32Δ (CC845-1A) strains were plated onto YNB minimal medium in the presence and absence of 20 μM Cd²⁺ and grown for 2 days.

Figure 7

Model for Cd²⁺-mediated activation of the MET gene network through the inhibition of the SCF^Met30 ubiquitin ligase and activation of an uncharacterized deubiquitinylating enzyme (Ubp).