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Figure 4


Fig. 4. 19S RP and base compete with each other for binding to the ER. (A) Upper panel: schematic representation of 26S proteasome subparticles. Positions of FLAG tags for purification are indicated; the Pre1p-FLAG strain was used for purification of 20S particles, all other particles were purified from the Rpt1p-FLAG strain RJD1171. Lower panel: Coomassie Blue-stained gel of purified 26S proteasomes and subparticles. Note the slight contamination of the lid fraction with intact 19S RP. (B) Dog PK-RM (10 eq) were incubated with 5 pmol lid or 1 pmol base and analyzed by flotation in a sucrose gradient, followed by SDS-PAGE and immunoblotting for Rpn12 (lid) or Rpt5p (base). (C) Dog PK-RM (10 eq) were incubated with 2.5 pmol base in the absence or presence of 10x excess 19S RP, and analyzed as in B. Base binding was detected using the anti-FLAG antibody, 19S RP binding with anti-Rpn12 antibody, and quantified using chemiluminescence and a CCD camera system (Raytest, Germany).





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