Figure 6. Ubiquitinated Rpb1 accumulates on chromatin in UV-treated cdc48-3 mutant.
(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