(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.

(A) WT (W303 and 204) and mutant cells were shifted to 37°C for 90 minutes and UV irradiated. Lysates were fractionated on a GstDsk2 resin. The input extract and bound fractions were immunoblotted (IB) for Rpb1 (4H8) and Ub, as indicated. 204 is RJD487, a wild-type congenic to cdc43-2, and untreated sample and beads were partially lost during IP.
(B) Rpb1 Ub conjugates accumulated in ubx5Δ but not ubx1Δ, and were diminished in cul3Δ mutant cells. Methods same as (A) except that cells were maintained at 30°C. Asterisk indicates sumoylated Rpb1.
(C) Rpb1 Ub conjugates hyperaccumulated in a ubx4Δubx5Δ double mutan. Methods same as (A) except that cells were maintained at 30°C.
(D) Ub conjugates accumulated on proteasomes isolated from ubx4Δ and ubx5Δ mutants. Lysates from wildtype and mutant cells expressing Flag-tagged Pre1 (or not) were immunoprecipitated (IP) with anti-Flag and immunoblotted (IB) for Ub, Rpn11 and 20S. Input extracts were blotted for 20S.
See also Figure S5.

(A) CUL3-TAP (RJD4680) cells were treated with UV or not, and harvested after the addition of 0.1% azide. Spheroplasts were prepared using 25 units of Zymolyase 100T for 2 × 10⁹ cells. The three fractions: Whole cell extract (WCE), supernatant (Sup), and chromatin pellet (ChrPell) derived from spheroplasts were immunoblotted (IB) with the indicated antibodies.
(B) Cdc48 and 26S proteasome recruitment to chromatin was enhanced following UV. Crude chromatin pellets (Frc3) obtained by centrifuging WCE (Frc1) through a sucrose cushion were fractionated further following limited digestion with micrococcal nuclease (MNase) to generate high-speed pellet (Frc6) and supernatant (Frc7) fractions. Frc6 is enriched in released polynucleosomes. All fractions were immunoblotted (IB) for histone H3, 19S (Rpn3), 20S proteasome subunits, and Cdc48. Fraction numbers correspond to lane numbers.
(C) Ubiquitinated Rpb1 accumulated on chromatin in response to UV and inactivation of Cdc48. WT and mutant cells expressing Myc-tagged Ub were shifted to 37°C, and Cu2+ was added to induce expression of Ub. After two hours, cultures were irradiated with UV (or not) and harvested. Crude chromatin pellets (FrC) were isolated as in (B) above and treated with Benzonase. Solubilized material was immunoprecipitated (IP) with anti-myc, and aliquots were blotted (IB) for Rpb1 (4H8), Spt5, and Rpn3.
(D) Cells of the indicated genotype were galactose-induced for two hours, then treated (or not) with MG132 for 30 min, lysed, and subjected to immunoprecipitation with anti-myc. The resulting samples were immunoblotted with antibodies to detect the indicated antigens. pGST-UBX5 is a plasmid that expresses GST-tagged Ubx5 under the GAL promoter.
See also Figure S6

(A) Ub conjugates accumulated on proteasomes in cdc48-3. Wildtype (WT; RJD3437) and mutant (RJD3454) cells expressing myc-tagged Pre1 (or not, RJD4090) were shifted to 37°C for 90 min and treated with 40 µM MG132 for 30 min. Proteasomes were isolated by anti-myc immunoprecipitation (IP) and immunoblotted (IB) for Ub and Pre1-myc (upper panels). Input extracts were blotted for Ub and Rpn3 (lower panels).
(B) Accumulation of conjugates on proteasome was not due to a defect in chymotryptic activity in cdc48-3 mutants. Isolated 26S proteasomes fractionated by native PAGE were either stained with Coomassie Blue (CB) or processed for in-gel peptidase activity using the fluorescent reporter substrate LLVY-AMC. R1C and R2C refer to 20S complexes capped by 1 or 2 19S regulatory particles, respectively.
(C) Numerous proteins accumulated on proteasomes in cdc48-3 mutants. Multidimensional mass spectrometry (MudPIT) was performed on an LTQ mass spectrometer using 26S proteasomes isolated from WT and mutant (RJD2902) cells grown at 37°C for 2 hours. The actual ratio of total spectral counts of proteasomal subunits from mutant to WT was 0.8 and was normalized to one. The same normalization factor was applied for spectral counts obtained for the proteasome-interacting proteins (PIPs). ∞ refers to proteins found in cdc48-3 but not WT proteasomes.
(D) Rpb1 accumulated on proteasome in cdc48-3 mutant treated with UV. WT and mutant cells were shifted to 37°C for 90 minutes and UV irradiated. Cells were recovered at 37°C for 30 minutes in the absence or presence of MG132. Input cell extracts and proteasomes immunoprecipitated (IP) with anti-myc were blotted (IB) for Rpb1 using 4H8 antibody.
See also Figure S1.

(A) Npl4 and Ufd1 contributed to Rpb1 turnover. Wildtype and mutant cells (npl4-1/RJD2589 maintained at 30°C, and ufd1-2/RJD3264 shifted to 37°C for one hour) were UV irradiated at 0 min. Cultures were processed as described in Figure 2C.
(B) Ubx1 and Ubx5 contributed to Rpb1 turnover. WT (S288C) and mutant cells (RJD4614, 2551, 3176, 3177) maintained at 30°C were irradiated with UV at 0 min. Cultures were processed as described in Figure 2C. Results are presented as means ± SEM of three independent experiments.
(C) Ubx4 contributed to Rpb1 turnover. WT and mutant cells (RJD5249, RJD5246, RJD5247, RJD5248) maintained at 30°C were irradiated with UV at 0 min. Cultures were processed as described in Figure 2C.
(D) Increased impairment of Rpb1 turnover in ubx4Δubx5Δ double mutants compared to single mutants. WT and mutant cells (RJD5259) maintained at 30°C were irradiated with UV at 0 min. Cultures were processed as described in Figure 2C.

(A) UV irradiation stimulated interaction of Ubx5 with Rpb1. Untagged (RJD4614), and UBX5-MYC (RJD4214) strains were UV irradiated or not, and lysates were immunoprecipitated (IP) with anti-myc antibody. Aliquots were blotted (IB) for Cdc48 and Rpb1 (using 4H8). MG: +MG132.
(B) Rpb1 accumulated on 26S proteasomes isolated from ubx5Δ cells. Cells expressing HBH-tagged Rpt5 (RJD4741 and RJD4742) were irradiated with UV and crosslinked with formaldehyde. Lysates were prepared in 6M guanidine-HCl and 26S proteasomes isolated by consecutive Ni2+-NTA and streptavidin (SA) affinity chromatography under denaturing conditions. Purified samples were blotted (IB) for Rpb1 (4H8), Rpt5-HBH (anti-His), and 20S (anti-a7).
(C) Ub conjugates that accumulated in ubx5Δ cells were substantially dependent on Cul3 but not Rsp5. Methods as in Figures 4A, B.
(D) Ubx5 and Cul3 interacted. Cells expressing HA-tagged Cul3 from a plasmid and Myc-tagged Ubx5 were irradiated with UV, or not. Input extracts and anti-myc immunoprecipitates (IP) were analyzed for their content of Cul3 by immunoblotting (IB) with anti-HA.

The Rpb1 subunit of RNA Pol II holoenzyme (H) irreversibly stalled at sites of DNA lesions is ubiquitinated by the Cul3–RING ligase complex. UbRpb1 can independently recruit proteasome and Ubx5-Cdc48 complexes. UbRpb1 is extracted from its binding partners in an unfolding reaction dependent on Ubx4 or Ubx5-Cdc48 and is threaded into the 26S proteasome.