FIG. 2.

Turnover of an Ste3-Ub fusion protein in the endocytosis-defective mutants. A 2-h period of GAL1-driven expression of Ste3-Ub (A) or Ste3p (B) was induced in wild-type (wt) or end mutant yeast cultures with galactose addition and terminated with glucose addition. Fifteen minutes prior to glucose addition, cultures were shifted from 25 to 37°C. Coincident with the glucose addition (0-h time point) and at the indicated glucose chase time points, culture aliquots were removed and treated with energy poisons (see Materials and Methods). Extracts were then prepared, and the loss of the Ste3 antigen was monitored by Western blotting with Ste3p-specific antibodies. (A) Ste3-Ub turnover. Wild-type and end mutant MATα GAL1-STE3-UB strains from two different strain backgrounds were used. Strains in the end4-1 background (top panel) included wild-type (NDY990), end4-1 (NDY991), pep4Δ (NDY992), akr1Δ (NDY1011), vrp1Δ (NDY1042), and end3Δ (NDY1076) strains. NDY343 (ste3Δ) was included as control for the specificity of Ste3p antibodies. Strains in the yck1Δ yck2^ts background (bottom panel) included wild-type (NDY1012), end3Δ (NDY1074), yck1Δ yck2^ts (NDY1090), akr1Δ (NDY1085), akr1Δ pep4Δ (NDY1218), and yck1Δ yck2^ts pep4Δ (NDY1223) strains. The high level of Ste3-Ub protein apparent in NDY992 cells (pep4Δ) likely reflects the augmented growth rate of this strain relative to the isogenic wild-type strain seen in raffinose- and/or galactose-containing culture media, not to the pep4 turnover blockade: the end mutants in which turnover is blocked (e.g., end3, end4, vrp1, yck^ts, and akr1 mutants) do not have similar levels of Ste3-Ub protein over accumulation. (B) Ste3p turnover in wild-type and end mutant cells. Four isogenic MATα GAL1-STE3 strains were used for this analysis, i.e., wild-type (NDY877), yck1Δ yck2^ts (NDY913), akr1Δ (NDY1083), and end3Δ (NDY1118) strains.