Ddi1, Dsk2, and Ub conjugate recruitment to the proteasome is compromised in rpn1-D517A and rpn1-K484A. (A) Affinity-purified rpn13Δ rpn1-K484A and rpn13Δ rpn1-D517A proteasomes contain reduced levels of Dsk2. Detergent was present during the binding step of the anti-Flag immunoprecipiation as described in the Methods section. (B) Affinity-purified rpn13Δ rpn1-D517A proteasomes contain reduced levels of Ddi1 and Ub conjugates. Levels of UBA-UBL proteins, the lid subunit Rpn12 and polyubiquitin are shown for affinity purified proteasomes (IP) and in the whole cell extract input (WCE). This purification was performed in the absence of detergent. Densitometric quantification of the blot is shown (right panel). The amount of UBL protein was normalized to Rpn11FLAG and wild type levels were set as 100%. (C) Proteasomes isolated from rpn1-D517A are intact. SDS-PAGE and native gel analysis of affinity purified 26S proteasomes from Rpn11-Flag tagged strains. The native gel was incubated with Suc-LLVY-AMC in the presence of ATP and 0.05% SDS to visualize RP and CP activity. The isoforms of the 26S proteasome are indicated. (D) Quantitative SILAC isotopic ratios are shown for all subunits of the proteasome isolated from an rpn13Δ strain (labeled with heavy isotopes; "H") in comparison to proteasomes isolated from an rpn13Δ rpn1-D517A strain (labeled with light isotopes; "L").