Figure 6.

Two functional units within the base. (a,b) Isolated 26S proteasome holoenzymes were fractionated along a phosphate gradient. Eluted fractions were resolved by SDS-PAGE and stained for protein (a) or immunoblotted for proteasome subunits (b). Proteasomes of specific subunit composition concentrate into separate fractions. Similar fractionation was observed for the open-channel mutant (Supplementary Fig. 7 online). (c) The isolated complex from fraction 16 was subjected to MS/MS analysis. Two protein bands migrating slightly higher than 100 kDa were identified by MS/MS as Rpn1 and Rpn2. No peptides derived from any other Rpn or Rpt subunit were detected in the sample. (d) AFM images of representative particles identified in each fraction (fr.): 14, proteasomes sans lid (Base–CP complexes); 15–16, the newly identified 20S-extended species (Rpn1-Rpn2–CP, and Rpn2–CP); 17, stripped core particle (20S CP). Above, top views and horizontal sections. Below, the predominant isolated species are indistinguishable from reconstituted complexes of defined composition. (e) Peptidase activity of proteasome subspecies from each fraction (taken from a,b) with and without 0.02% (w/v) SDS. The change relative to basal levels is shown as a bar graph for each fraction. (f) The rate of casein proteolysis by proteasomes from each fraction was measured. Error bars represent s.d. of three independent experiments.