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. 2020 Feb 7;16(2):e1008597. doi: 10.1371/journal.pgen.1008597

Fig 5. Met30 and Cdc4 interact in vivo and cooperatively regulate proteolysis of Cse4.

Fig 5

(A) Cdc4 and Met30 cooperatively regulate proteolysis of Cse4. Western blot analysis was performed with WCE prepared from WT (YMB9673), met30-6 (YMB8789), cdc4-1 (YMB9571) and cdc4-1 met30-1 (YMB10033) strains expressing endogenous HA-Cse4. The percentage of remaining HA-Cse4 at 90 minutes after CHX treatment (50 μg/ml) is indicated. Results from two biological experiments are shown as mean ± average deviation. (B) Met30 interacts with Cdc4 in vivo. Top panel: Co-IP was performed with anti-HA antibody using WCE from cdc4Δ::HA-CDC4 strain (YMB10217) with Myc-MET30 (pK699); control strains WT (BY4741) with either vector (pRS415) or Myc-MET30 (pK699) grown in selective glucose medium at 25°C. Western blot analysis of Input and IP (anti-HA) samples were analyzed using anti-HA and anti-Myc antibodies. Bottom Panel: Co-IP was performed with anti-Myc using WCE from cdc4Δ::HA-CDC4 strain (YMB10217) with Myc-MET30 (pK699); and control strains (YMB10217) with vector (pRS415) grown at 25°C. Western blot analysis of Input and IP (anti-HA) samples were analyzed using anti-HA and anti-Myc antibodies. All tagged proteins are expressed from their native promoter. (C) Schematic of Met30 domains. Homodimerization domain (D), F-box and WD40 domain with amino acid numbers indicated. (D) The N-terminus, homodimerization domain (D domain) and F-box of Met30 are dispensable for the interaction of Met30 and Cdc4. Co-IP experiments were performed with anti-HA using WCE from a cdc4Δ::HA-CDC4 strain (YMB10217) with Myc-MET30 (pK699), Myc-met30ΔF-box (Δ187–227 aa, pK680), Myc-met30Δ77 (Δ1–77 aa, pMB1835), Myc-met30Δ113 (Δ1–113 aa, pMB1837) or Myc-met30ΔD (Δ124–182 aa, pMB1830) and control WT strain (BY4741) with Myc-MET30 (pK699) or Myc-met30ΔD (Δ124–182 aa, pMB1830) grown at 25°C. Western blot analysis of Input and IP (anti-HA) samples were probed with anti-Myc and anti-HA antibodies. All tagged proteins are expressed from their native promoters. (E) The WD40 domain of Met30 is required for the interaction of Met30 and Cdc4. Co-IP experiments were performed with anti-HA using WCE from a cdc4Δ::HA-CDC4 strain (YMB10217) with Myc-MET30 (pK699) or Myc-met30ΔWD40 (Δ277–640 aa, pMB1861) and control WT strain (BY4741) with Myc-MET30 (pK699) or Myc-met30ΔWD40 (Δ277–640 aa, pMB1861) grown at 25°C. Western blot analysis of Input and IP (anti-HA) samples were analyzed using anti-HA and anti-Myc antibodies. All tagged proteins are expressed from their native promoters.