Fig. 4: Stabilization of Hcm1 partially mitigates the effects of TAD phosphorylation.

a–c Strains expressing the indicated HCM1 alleles from plasmids were co-cultured and the percentage of cells expressing each allele was determined at the timepoints indicated. In total, 5000 cells were analyzed from each timepoint. Shown is an average of n = 3 experiments, error bars represent standard deviations. Black represents cells expressing WT HCM1 (a), purple represents cells expressing hcm1-3N (a–c), blue represents cells expressing hcm1-3N8A (b), and red represents cells expressing hcm1-3N8E (c). d Schematic showing the design of the 3N A/E phosphomutant library. D indicates the phosphodegron consisting of three S/T-P sites that have been mutated to A-P (3N). The eight S/T-P sites in the TAD have been mutated to either A-P or E-E in all 256 possible combinations. e, f Box and whisker plots showing the median selection coefficients of groups of mutants with the indicated number of phosphomimetic mutations (e), or with specific substitutions at the indicated positions (f). Color gradient from blue (hcm1-3N8A) to red (hcm1-3N8E) corresponds to increasing numbers of phosphomimetic mutations within the Hcm1 TAD (e), Blue corresponds to all mutants with A-P at the indicated site, red corresponds to all mutants with E-E at the indicated site (f). The black center line indicates the median, boxes indicate the 25th−75th percentiles, black lines represent 1.5 IQR of the 25th and 75th percentile, black circles represent outliers. Shown is all data from n = 3 biological replicates. Supplementary Fig. 3b shows correlations between replicates; Supplementary Fig. 5 shows elevated expression of Hcm1-3N and heatmaps comparing A/E and 3N A/E screens. g Cumulative frequency plot showing the cumulative fraction of mutants that display selection coefficients that are less than or equal to the indicated values. Black represents the A/E library, purple represents the 3N A/E library. Note that selection coefficients in the A/E library are calculated with relative to WT HCM1 and selection coefficients in the 3N A/E library are calculated relative to hcm1-3N. Shown is data from n = 3 biological replicates.