Skip to main content
. Author manuscript; available in PMC: 2011 Jul 7.
Published in final edited form as: Mol Cell. 2011 Jan 7;41(1):82–92. doi: 10.1016/j.molcel.2010.12.017

Figure 2. The ATPase activity of Cdc48 is required for UV-induced turnover of the largest subunit of RNA Polymerase II, Rpb1.

Figure 2

(A) Rpb1 was stabilized in cdc48-3 cells irradiated with UV. WT (RJD360) and mutant (RJD3411) cells were shifted to 37°C for one hour, irradiated with UV, and transferred to medium containing 100 µg/ml cycloheximide. At the indicated time-points, cells were harvested and lysed for immunoblot (IB) analysis of Rpb1 using 8WG antibody. Tubulin served as the loading control.

(B) WT, but not ATPase-deficient Cdc48, restored Rpb1 turnover in UV-irradiated cdc48-3 cells. Mutant cdc48-3 (RJD3411) cells containing plasmid-borne WT GAL-CDC48His6 (RJD4996) or the Q2 mutant (RJD4997) were grown in galactose for two hours to induce expression of ectopic Cdc48. Induced cells were shifted to 35°C (the minimum restrictive temperatue in this medium) for one hour, UV irradiated, and then sampled at the indicated time-points.

(C) Stabilization of Rpb1 in cdc48-3 was not suppressed by rad26Δ. Single (RJD3411 and 4523) and double (RJD4570) mutants were shifted to 37°C for one hour, UV irradiated, and processed as above. Quantification was performed on LI-COR Odyssey with normalization to tubulin. Following quantification, all data were plotted using Prism software on a logarithmic scale on the y-axis.

(D) Cdc48 associated with Rpb1. Cells were UV treated or not and lysates prepared for immunoprecipitation (IP). Aliquots of inputs and immunoprecipitates (IP) were analyzed for their content of Cdc48 and Rpb1 by blotting (IB) with TAP and 4H8 antibodies, respectively.

See also Figure S3.