Figure 4. Est1p degradation in G1 phase requires three destruction boxes (D-boxes).

(A) Schematic of EST1 shown to scale. EST1 contains six putative D-boxes with sequence RxxL (boxes labeled 1–6). Deletion of the C-terminal 300 amino acids (CΔ300) results in a truncated protein that removes putative D-boxes 5 and 6. The N-terminal 52 amino acids are shown, with putative D-boxes 1 and 2 outlined. Upward pointing black triangles represent the position of the indicated N-terminal deletion. (B) D-boxes 1, 2, and 4 contribute to Est1p degradation. YKF802 containing pKF600 (GAL1-HA₃-EST1) plasmids expressing either wild-type EST1 (WT) or the D-box (DB) mutated (RxxL to AxxA) est1 alleles indicated were treated as in Figure 1B, except strains were arrested with α-factor. Bars represent the average HA₃-Est1p half-life for three independent biological replicates; error bars are the standard deviation of the mean. Using a two-tailed t-test, there is no significant difference from WT for D-box 3 (p-value 0.833) or D-boxes 5+6 (p-value 0.104). D-box 1 (p-value 0.027), D-box 2 (p-value 0.012), D-boxes 1+2 (p-value 0.001), D-box 4 (p-value 0.001) and D-boxes 3+4 (p-value 0.002) are significantly different than WT, denoted by *. (C) Deletion of D-box 1 or 2 stabilizes Est1p during G1 phase. YKF802 containing pKF600 plasmids expressing either wild-type EST1 (WT) or the est1 deletion variants indicated (CΔ300, NΔ7, NΔ15, NΔ25, NΔ35 or NΔ50) were treated as in (A). Bars represent the average HA₃-Est1p half-life for independent biological replicates: n = 3 for each variant except NΔ50, where n = 4. Error bars are standard deviation from the mean; significance is denoted by *. By a two-tailed t-test, there is no significant difference between WT and CΔ300 (p-value 0.445) or NΔ7 (p-values 0.188). The half-lives observed for NΔ15 (p-value 0.0003), NΔ25 (p-value 0.008), NΔ35 (p-value 0.005) and NΔ50 (p-value 0.02) are significantly different from WT.