The Mkt1-G30D point mutation decreases Puf3-dependent protein expression. (A) Western blot reflecting Cox2, Mkt1-Flag, and Por1 protein levels in CEN.PK cells during growth in YPD and 3 h after switch to YPL medium. Numerical values represent relative densities of Cox2 bands compared with corresponding G6PDH bands. Note: Endogenous substitution of Mkt1-D30 alleles diminished Mkt1-Flag and Cox2 levels. (B) Growth curves of indicated strains monitored in an automated plate reader during incubation at 30°C in YPL medium. OD₆₀₀ measurements were taken every 30 min. CEN.PK Mkt1-D30 cells exhibited reduced growth compared with WT and Mkt1-G30 cells. Traces are from one representative experiment (n = 3 per group). (C) Western blot depicting Puf3-HA levels in CEN.PK cells grown in YPD to log phase and 5 h following switch to YPL medium. CEN.PK Mkt1-D30 cells had reduced Puf3-HA protein accumulation in YPL medium compared with WT and Mkt1-G30 cells. (D) Western blot depicting Cox2, Mkt1-Flag, Pab1, and Por1 protein levels from CEN.PK and S288C cells grown to log-phase in YPD and 5 h following switch to YPL medium. Endogenous substitution of Mkt1-G30 alleles rescued Cox2 and Mkt1-Flag protein levels in the S288C strain background. (E) Growth curves of CEN.PK and S288C cells during incubation in YPD medium obtained using the method described in B. All strains grew at similar rates (n = 3 per group). (F) Growth curves of CEN.PK and S288C cells during incubation in YPL medium obtained using the same method described in B. Endogenous substitution of Mkt1-G30 alleles in S288C yeast enhanced growth in YPL medium (n = 3 per group). (G) Western blot depicting Puf3-HA levels in CEN.PK and S288C cells grown in YPD and 5 h following switch to YPL medium. Puf3-HA levels in cells collected from YPL medium were reduced in S288C WT and Mkt1-D30 cells and rescued in S288C Mkt1-G30 cells. Source data are available for this figure: SourceData F8.