Figure 1.

Affinity purification of 26S proteasomes, plus 19S and 20S subcomplexes. (A) Extracts of yeast strains expressing Pre1^FH (RJD 1144, lanes 2 and 4) or Rpt1^FH (RJD 1171, lane 3) were prepared and bound to anti-Flag M2 resin as described in MATERIALS AND METHODS. Bound proteins were eluted with 100 μg/ml Flag peptide and analyzed by SDS-PAGE and Coomassie blue staining. The entire purification from the PRE1^FH strain was carried out either in the presence (lane 2) or absence (lane 4) of ATP. The purification from the RPT1^FH strain was carried out in the presence of ATP (lane 3). Lane 1 depicts a control purification performed with extract from an untagged strain (RJD 487) in the absence of ATP. (B) 26S proteasomes, 19S caps, and mock samples were prepared as described in A from RJD 1144, RJD 1171, and RJD 487, respectively, either in the absence of ATP or in the presence of ATP or ATP-γ-S, as indicated in the figure. Purified samples were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with polyclonal antisera against the mammalian 20S subunit LMP7, which cross-reacts with budding yeast Pre2/Doa3, and the budding yeast 19S subunits Rpn10 and Rpt1.