FIG. 3.
Ime2p-6XHA is stabilized in the grr1Δ mutant. (A) Shutoff of transcription from the tetO promoter occurs normally in the grr1Δ mutant. Wild-type (WT) or grr1Δ strains expressing the tetO-IME2-6XHA allele (SH2811 and 2813, respectively) were transferred to sporulation medium containing tetracycline, samples were removed at the indicated times (0 to 75 min), and IME1, IME2, and a control transcript (Cntrl) were analyzed as described for Fig. 1. (B) Ime2p-6XHA levels in Sp medium. For the cultures shown in panel A, samples were removed at the indicated times (0 to 90 min) and Ime2p-6XHA levels and Cdc28p were analyzed by Western blotting. (C) Graph representing Ime2p-6XHA expression relative to Cdc28p expression in wild-type (triangles) and grr1Δ (circles) strain from three independent trials of the experiment shown in panel B. (D) Ime2p-6XHA stability in Sp+0.5 medium. Cultures were grown and analyzed as described for panel B. (E) Graph representing Ime2p-6XHA expression relative to Cdc28p expression in wild-type (triangles) and grr1Δ (circles) strains for three independent trials of the experiment shown in panel D. Error bars in panels C and E represent the standard errors of the means of three independent experiments. (F) Strains expressing both Ime2p-6XHA and myc-Ubi(KO) were incubated in Sp+0.5 medium for 6 h, immunoprecipitated with anti-HA antibodies, and analyzed for both Ime2p-6XHA and myc-Ubi(KO)-conjugated proteins by Western blotting. The arrow points to the expected position of unmodified Ime2p-6XHA on the anti-Myc blot based on comparison of this blot with the anti-HA blot. (G) Ime2p-6XHA stability in Sp+0.5, analyzed as described in the legend for panel B, for wild-type (WT), cdc34-2, and cdc53-1 strains at 37°C (SH3183, SH3179, and SH3181, respectively).