a, Proteasomes were affinity-purified from strains expressing HA-tagged Nas6. Samples were resolved by SDS-PAGE and immunoblotted for Nas6 (anti-HA), RP (anti-Rpn8 and anti-Rpn12) and CP (anti-α7).
b, Proteasomes were affinity-purified from strains expressing HA-tagged Hsm3, eluted, and analyzed for the presence of Hsm3, RP, and CP.
c, Proteasomes were affinity-purified from strains expressing HA-tagged Nas6 and Rpn14. After elution, samples were analyzed as in a.
d, HA-specific immunoprecipitations were tested for RP and CP by immunoblotting.

a, RP, lid, base bound to IgG beads or untreated IgG beads were incubated with GST-Nas6, GST-Rpn14, or GST-Hsm3 purified from E. coli. Beads were analyzed for bound proteins.
b, Domain composition of Rpt3; other Rpt proteins have similar architecture. “cc” predicted coiled-coil region; “ATPase” ATPase domain; “C” C-domain20; “aa” amino acids.
c, His-tagged C-domains were co-expressed with GST or GST-tagged Nas6, Rpn14, or Hsm3 in E. coli. Glutathione-Sepharose purified samples were immunoblotted using His-tag antibody followed by Coomassie Blue staining.
d, Modeling of the interaction of Rpt ring with CP in the presence (right) and absence (left) of Nas6: Nas6 binding to the ATPase ring appears to block the C-terminal tail of Rpt3 from docking into the CP. The CP α-ring (beige)24, was combined with the PAN hexameric ATPase23 (ATPase domain grey; C-domains blue). The Nas6-Rpt3 structure21 was mapped onto the ATPase ring by aligning the Rpt3 structure with the PAN C-domain (dark blue; C-terminus red), with R.M.S. 1.1 Å over 283 atoms. The last twelve residues of the PA26 C-terminus17 (magenta) are docked into CP17–19. The length corresponds to that of Rpt3 C-terminal tail that is absent from the Nas6-Rpt3 structure.
e, Affinity-purified, resin-bound base was incubated with indicated RP-chaperone, washed, then loaded with purified CP. Resin-bound proteins were eluted and analyzed by immunoblotting.

a, Strains with indicated genes deleted were spotted on plates in four-fold dilutions and grown at the indicated temperature.
b, Cell lysates (75µg) were resolved on native gels. Gels were stained for LLVY-AMC hydrolytic activity.
c, Cell lysates of wild-type or nas6Δ rpn14Δ strains were resolved by two-dimensional native-SDS/PAGE gel electrophoresis followed by immunoblotting.
d, Cell lysates were resolved on 5.25% native gels and immunoblotted. BP1 assignment based on ref. 2. *, background band.

a, nas6Δ rpn14Δ strains expressing NAS6 or gankyrin from a GAL promoter were spotted onto YPD plates or YEP Raf/Gal plates in four-fold dilutions, and incubated at indicated temperatures.
b, Proteasomes were purified from yeast strains expressing HA-tagged gankyrin and analyzed by immunoblotting.
c, His-tagged C-domains of indicated human Rpt proteins were co-expressed in E. coli with GST-tagged gankyrin. Glutathione-Sepharose purified samples were separated by SDS-PAGE, and stained with Coomassie Blue. Band assignments were confirmed by immunoblotting (data not shown).
d, Human proteasomes purified via HTBH-tagged Rpn11 (Ref. 5) were were resolved by two-dimensional native-SDS/PAGE gel electrophoresis. Proteins were silver stained or analyzed by immunoblotting.
e, as c, only GST-gankyrin was replaced by GST-S5b.
f, HeLa cell lysate was resolved on Blue native gels. Fractions were analyzed by mass spectrometry, and protein abundance estimated using spectral counting. Based on their abundance profiles, most proteasome subunits can be divided into four groups (left graph); CP (α1–7 and β1–7), lid (Rpn3,5,6,7,8,9,10,11), baseB (Rpt3,4,5,6 and Rpn2) and BaseA (Rpt1,2, and Rpn1). Interestingly, S5b is absent from proteasomes (fraction 1 and 2), but abundant in fraction 4. Right panel, S5b and individual profiles of BaseA members.
g, HeLa cell lysates from e were separated by 2D native-SDS-PAGE and analyzed by immunoblotting.