(A) Stably purified lid intermediates from Figure 4(B) were incubated with purified base-CP (equivalent to lidless proteasome) and resulting association monitored by proteolytic assay on native gel. (B) Specific interactions of Rpn11 with RPT ATPases depends on C-terminal fragment. Each RPT ATPase was purified and immobilized on CH-sepharose beads and incubated with either full-length Rpn11 or Rpn11ΔC. Beads with immobilized BSA were used as a negative control (mock). Bound proteins were separated on SDS/PAGE and immunoblotted with anti-Rpn11. (C) Assembly pathway of lid module 1. Relative orientation of subunits is based on PDB 4CR2. (D) Module 1 can serve as a de facto lid core. A summary of proteasome species identified in the present study, from left to right: 1. base-CP, 2. module1–base-CP, 3. incomplete 26S identified in rpn11–m1 containing a lid core, 4. proteasomes from rpn11–m1 upon addback of Rpn11 C-terminal fragment and 5. 26S proteasome holoenzymes. Relative orientation of lid subunits running along the side of the base (illustrated as a pink mound) is based on PDB 4CR2. Schematic depiction of main sub-complexes and key lid subunits as follows: brown cylinder, 20S CP; pink mound, base; blue, lid core (module 1) subunits; red, labile (module 2) lid subunits; violet, Rpn10 bridging lid and base.