Fig. 1: Grr1 is required for tunicamycin resistance, Hac1p expression and Ubp3 degradation in ER stress conditions.

A grr1∆ and pep3∆ strains are sensitive to tunicamycin (Tm). Yeast cells lacking one of the E3 ligases and strains transformed with plasmids encoding each wild type were grown in SDC-Leu medium. 10-fold serial dilutions of OD₆₀₀ = 0.3 cells were prepared in the 1.5 mL tubes and spotted onto Tm-added SDC-Leu and control plates. Plates were incubated at 30 °C for 2–3 days. B Hac1p expression is decreased in grr1∆ strains. Yeast cells were grown at 30 °C until OD₆₀₀ = 0.2, then treated with 1 µg/mL of Tm for ~4 h and harvested. The samples were analyzed using western blotting with anti-Hac1 antibody. CBB staining was used as a loading control. C The splicing of HAC1 mRNA occurs in grr1∆ and pep3∆ cells. Yeast cells were grown and harvested as in (B). The samples were analyzed by northern blotting with DIG-labeled HAC1 probe. Methylene Blue staining was used as a loading control. D Ubp3 downregulation is suppressed in the grr1∆ cells. The indicated yeast cells expressing UBP3-3HA were grown and harvested as in (B). The samples were analyzed using western blotting with anti-HA and anti-α-Tubulin antibodies. E, F Grr1-dependent rapid decay of Ubp3 under ER stress conditions. The indicated yeast cells expressing UBP3-3HA were grown at 30 °C until OD₆₀₀ = 0.2, then treated with 1 µg/mL of Tm for 1 h. Yeast cells were then treated with 0.25 mg/mL of cycloheximide (CHX) and harvested at the indicated times. The samples were analyzed with western blotting using anti-HA antibodies. CBB staining was used as a loading control. Dilutions are 100%, 75%, 50%, 25%, 12.5%, 6.25%. G The half-life of Ubp3-3HA in the wild-type and the grr1∆ mutant cells. The Ubp3-3HA/CBB levels shown in (E, F), Supplementary Fig. 2A, B were quantified and normalized relative to that at 0 min samples. Data represent n = 3 biologically independent experiments (mean ± SE), and P values were calculated by Two-sided Welch’s t-test. All experiments were repeated at least twice with biologically independent samples and showed similar results. Source data are provided as a Source Data file.