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

Fig 3. Met30 and Cdc4 interact with Cse4 and regulate ubiquitin-mediated proteolysis of Cse4.

Fig 3

(A) Increased stability of overexpressed Cse4 in met30-6 and cdc4-1 strains. Western blot analysis was performed with whole cell extracts (WCE) prepared from strains expressing GAL-HA-CSE4 (pMB1597) grown in galactose media for one hour for WT strain and 3 hours for met30-6 (TSA848) and cdc4-1 (TSA878) strains at 25°C and probed with anti-HA (HA-Cse4) and anti-Tub2 antibodies (loading control). Percentage of remaining HA-Cse4 (normalized to Tub2) at the 90 minutes after CHX treatment is shown. Results from three biological repeats are shown as mean ± standard deviation. (B) Line graph for results shown in (A). (C) Reduced levels of ubiquitinated Cse4 in met30-6 and cdc4-1 strains. Ub pull-down was performed with WCE prepared after growth of strains in galactose medium for one hour for WT (BY4741) and three hours for met30-6 (YMB9353), and cdc4-1 (YMB9571) strains carrying vector or GAL-HA-CSE4 (pMB1597) at 25°C. WT strains expressing non-tagged Cse4 (empty vector) or HA- cse4(16KR) (pMB1892) were used as negative control for laddering pattern of ubiquitinated Cse4. Western blots were probed with anti-HA antibody. The percentage of ubiquitinated Cse4 is calculated by normalizing the amount of ubiquitinated Cse4 from the Ub pull-down to the levels of non-modified Cse4 in the input where WT is set to 100%. (D) cdc4-1 increases the stability of overexpressed Cse4 in quadruple mutant (psh1Δ slx5Δ rcy1Δ ubr1Δ)(YHR333) strain. The stability of overexpressed Cse4 (pMB1458) was examined in WT, quadruple (YMB11244) and quintuple (psh1Δ slx5Δ rcy1Δ ubr1Δ cdc4-1) (YMB11245) mutant strains. Growth in galactose medium was for two hours for WT and quadruple strains and three hours for the quintuple strain. The average of percentage of remaining HA-Cse4 from two biological repeats at 90 min post CHX treatment is shown. (E) Line graph for result shown in (D). (F) met30-6 further increases the stability of overexpressed Cse4 in psh1Δ strain. Stability of overexpressed Cse4 is determined as in (A) for WT(BY4741), met30-6 (YMB9353), psh1Δ (YMB9352) and met30-6 psh1Δ (YMB9350) strains. WCE prepared after growth of strains in galactose medium for one hour for WT and psh1Δ strains and 3 hours for met30-6 and met30-6 psh1Δ strains. The results represent the average of two biological repeats. A shorter (non-saturated) exposure of Western blot results for met30-6 psh1Δ is shown and used for quantification. (G) Line graph for results shown in (F). (H) Cse4 interacts with Met30 in vivo. Protein extracts from a WT strain (BY4741) expressing Myc-Met30 (pK699) with either vector (pMB433) or GAL-HA-CSE4 (pMB1597) were prepared after transient induction of Cse4 in galactose medium for 3 hours at 25°C. Input or IP (anti-HA) samples were analyzed by Western blot and probed with anti-Myc and anti-HA antibodies. For quantification, IP samples in two concentrations (undiluted and diluted 1:3) were loaded (indicated by the triangle). (I) Cse4 interacts with Cdc4 in vivo. Protein extracts from Myc-CDC4 strain (YMB9674) with either vector (pMB433) or GAL-HA-CSE4 (pMB1597) were prepared after transient induction of Cse4 in galactose medium for 3 hours at 25°C. Input or IP (anti-HA) samples were analyzed by Western blot and probed with anti-Myc and anti-HA antibodies.