FIGURE 6.

Deletion of the HbYX motif negatively affects proteasome assembly. A, immunoblotting analysis of affinity purified proteasome samples containing wild-type or mutant Rpts with the deletion of the HbYX motif. Cell lines that conditionally expressed FLAG-tagged wild-type or C-terminal three-residue deletion mutant Rpt subunits were established. Aliquots of FLAG-Rpt samples purified in the presence of 5 mm ATP were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. B and C, immunoblotting (B) and SDS-PAGE (C) analyses of affinity-purified FLAG-tagged wild-type or C-terminal deletion mutant Rpt3. The proteasome was affinity purified as described under “Experimental Procedures.” Ten microliters each of Rpt3 samples purified in the presence of 5 mm ATP was analyzed by immunoblotting with the indicated antibodies and by SDS-PAGE followed by Coomassie staining. D and E, immunoblotting (D) and SDS-PAGE (E) analyses of affinity-purified FLAG-tagged wild-type or C-terminal deletion mutant Rpt6.