Figure 6.
The Pib1 E3 ubiquitin-protein ligase targets Sec3-913 for degradation. A, growth on rich medium of wild-type, pib1Δ, sec3-913, and the double mutant was compared at the indicated temperatures. B, growth of the wild-type and sec3-913pib1Δ strains transformed with either an empty vector or a pib1+ expression vector was compared at the indicated temperatures. To select for the plasmids, these experiments were performed on EMM2 minimal medium. Note that we found that the sec3-913 strain was not quite as temperature-sensitive on minimal medium as on complete medium (e.g. in A). C, calcofluor staining of sec3-913 and sec3-913pib1Δ cells at 30 °C and 34 °C (left panel). Scale bar = 5 μm. The percentages of septated cells (septation index) and of septated cells with multiple septa were determined. The error bars indicate the standard deviation (n = 4). **, p < 0.01; ***, p < 0.001. D, the steady-state level of Sec3-913 in the indicated strains was compared by SDS-PAGE and Western blotting using antibodies to V5 (to detect Sec3-913) and, as a loading control, to α-tubulin. exp., exposure. E, the degradation of Sec3-913 protein was followed in sec3-913 and sec3-913pib1Δ cultures at 36 °C, where protein synthesis was inhibited with 100 mg/ml CHX for 4 h. Tubulin served as a control for equal loading. F, quantification of degradation experiments as in E; Sec3-913 degradation at 36 °C (dark gray, filled circles) and Sec3-913 at 36 °C in the pib1Δ background (light gray, filled squares). The error bars indicate the standard error of the mean (n = 4). G, the sec3-913-V5 strain carrying WT pib1 or a sec3-913-V5/pib1Δ strain transformed with a His6-tagged ubiquitin expression construct, as indicated, was treated with 1 mm bortezomib overnight, lysed, and used for precipitation (precip.) experiments with an Ni2+ resin in 8 m urea. The precipitated material was analyzed by blotting with antibodies to the V5 tag on Sec3-913 or to the His6 tag on ubiquitin. The arrowhead marks the position of non-ubiquitylated (Ubn) Sec3-913.