Article Figures & Data
Figures
- FIG. 1.
Effects of ela1Δ and cul3Δ mutations on UV sensitivity. (A) UV sensitivity of ela1Δ and cul3Δ strains. (B) UV sensitivity conferred by the ela1Δ and cul3Δ mutations in combination with the rad7Δ mutation. (C) UV sensitivity conferred by the ela1Δ and cul3Δ mutations in combination with the rad26Δ mutation. YPD plates containing 5 μl of serial 10-fold dilutions of exponentially growing yeast cells were UV irradiated and incubated in the dark at 30°C.
- FIG. 2.
Effects of elc1Δ, ela1Δ, and cul3Δ mutations on sensitivity to 4-NQO. (A) 4-NQO sensitivity conferred by the elc1Δ mutation in combination with the rad7Δ and rad26Δ mutations. (B) 4-NQO sensitivity conferred by the ela1Δ and cul3Δ mutations in combination with the rad7Δ mutation. (C) 4-NQO sensitivity conferred by the ela1Δ and cul3Δ mutations in combination with the rad26Δ mutation. Five microliters of serial 10-fold dilutions of yeast cells was spotted onto YPD plates containing the indicated amounts of 4-NQO and incubated at 30°C.
- FIG. 3.
Rpb1 is not degraded in UV- or 4-NQO-treated ela1Δ or cul3Δ cells. (A) Rpb1 levels in wild-type, ela1Δ, and cul3Δ strains following UV treatment. Yeast cell extracts were prepared at the indicated times after UV irradiation. Rpb1 and the loading control PGK1 were detected by SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with MAb 8WG16 and MAb PGK1, respectively. (B) Rpb1 levels in wild-type, ela1Δ, and cul3Δ strains following treatment with 4-NQO. 4-NQO was added to liquid cultures of log-phase yeast cells at the indicated concentrations, and cells were collected at 60 min after the addition. Cell extracts were prepared, and Rpb1 and PGK1 detected by Western blot analysis as performed for the cell extracts shown in panel A.
- FIG. 4.
Rpb1 polyubiquitylation does not occur in UV- or 4-NQO-treated ela1Δ and cul3Δ cells. (A) Absence of Rpb1 polyubiquitylation in UV-treated ela1Δ and cul3Δ cells. Yeast strains harboring plasmid YEp112, which expresses HA-tagged wild-type Ub, were grown to an OD600 of 1.0 in selective minimal medium, and Ub expression from the CUP1 promoter was induced with 100 μM CuSO4 for 2 h. After induction, cells in suspension were irradiated with UV (400 J/m2). Ubiquitylated proteins were purified with anti-HA resin (Sigma), separated by SDS-6% PAGE, and Rpb1 ubiquitylation analyzed with Rpb1-specific H14 antibody (Covance). (B) Absence of Rpb1 polyubiquitylation in 4-NQO-treated ela1Δ and cul3Δ cells. Methods were the same as described for panel A, except that cells were treated with 6 μg/ml of 4-NQO for 30 min. IP, immunoprecipitation; WB, Western blotting.
