20S proteasomes from β5proΔ β7^HC cells are similar to WT proteasomes. (A) Two-dimensional gel analysis of affinity-purified proteasomes. Cells expressed a β4 (PRE1) allele chromosomally tagged with Flag and His₆ epitopes (β4′). 20S proteasomes (30 μg) purified on an anti-Flag column from β5 β7^HC (MHY2831) and β5Δpro β7^HC (MHY2832) cells were subjected to isoelectric focusing in a pH range of 3–10, followed by SDS–PAGE and GelCode Blue staining. (B) Normalized peptidase activities of the three active centers (averages of two measurements). The nearly identical reduction for all sites relative to WT likely reflects a slightly lower concentration for the mutant particles.

Native gel analysis of affinity-purified proteasomal particles. (A) GelCode Blue-stained native gel showing affinity-purified protein complexes. Extracts from yeast β5 or β5Δpro cells with either Flag-His₆-tagged β4 (lanes 1 and 2) or β6 proteins (lanes 4 and 5) that also expressed β7 from a high-copy plasmid were purified on an anti-Flag affinity column. Control purification from untagged cells is shown in lane 3. (B) Native gel stained for protein (left) and assayed for peptidase activity by an activity overlay assay with the fluorogenic LLVY-AMC substrate (right). Conventionally purified 20S proteasomes (‘20S') were used for comparison. (C) Protein complexes affinity-purified with anti-Flag from β2-K29E β3-E142K cells and resolved on a GelCode Blue-stained native gel (first two lanes). For comparison, an anti-Flag purification from β5Δpro cells is also shown (last lane). (D) Pba1 and Pba2 bind directly to one another (GelCode Blue-stained SDS gel). T, total cell lysate; S, soluble fraction; P, pellet; F, Ni-NTA column flow-through; E, eluate from Ni-NTA. (E) Pba1 and Pba2 form a heterodimer. E. coli lysates expressing the following proteins were resolved by native PAGE (10%) and visualized with GelCode Blue: 1, Pba1-His; 2, Pba2-Flag; 3, Pba1-His+Pba2-Flag; 4, control lysate; 5, partially purified Pba1-His–Pba2-Flag complex. D, dimer; M, monomer.

High-copy β7^HC suppresses a specific β5 point mutant. (A) Suppression analysis of various β5 and β4 alleles by β7^HC. Top panel: mutant β5 (doa3/prg1) or β4 (pre1) or congenic WT strains were transformed with either empty vector or a high-copy β7 plasmid and then streaked on 1 μg/ml canavanine (30°C, 5 days). Lower panel: exponentially growing cultures were plated on YPD in 1:6 serial dilutions and incubated for 2 days at the indicated temperatures; the yeast strains were derived from MHY784 by transformation with the indicated plasmids followed by eviction of YCp50-DOA3 on FOA. (B) The β5 (doa3-1) mutant has an assembly defect that is partially rescued by β7^HC. Panels show anti-β5 and anti-β1 immunoblots of Superose-6 column fractions from extracts of doa3-1 cells that had been transformed with empty vector or a high-copy β7 plasmid. Anti-β1 antibody cross-reacts with an unknown protein (asterisk).

Accumulation of proteasomal subparticles in β5Δpro β7^HC cells. (A) Superose-6 chromatography followed by SDS–PAGE and anti-HA immunoblot analysis of whole-cell extracts from WT (MHY2498) and β5Δpro (MHY2506) cells. Alleles encoding β2-HA₂ and Ump1-HA₂ proteins replaced the corresponding endogenous loci. Peak positions of mature proteasomes, proteasome precursor complexes, and free subunits are indicated at the top of each panel. (B) Superose-6 column fractions resolved by nondenaturing PAGE with subsequent anti-HA immunoblotting. Proteasomes and proteasome subparticles are indicated at right (see Figure 3 legend). Ferritin (437 kDa) and thyroglobulin (669 kDa) were used as approximate size markers.

A β7_tail trans-ring interaction with β1 required for suppression of β5Δpro lethality. (A) Structure of the central proteasomal β rings (from PDB 1RYP) viewed down the dyad axis. Figure was prepared with RasMol 2.6. Arrowheads indicate the strictly conserved Trp211 residue (white) in the β7 tail. (B) Deletion of the β7 tail (residues 211–225; pre4-1) or mutation of Trp211 to Ala abolishes β7^HC suppression of β5Δpro. MHY1179 cells with the indicated YEplac181 plasmid-borne β7 alleles and pRS317 plasmid-borne β5 or β5Δpro were grown on fluoroorotic acid (FOA) medium (30°C, 5 days) to evict the WT β5 and β7 URA3-based plasmids originally present. (C) Mutation of β1 residues that disrupt the trans-interaction with the β7 tail impairs β7^HC suppression of β5Δpro. MHY1179 cells cotransformed with plasmids encoding the indicated β7 and β1 alleles (and also carrying a pRS317 plasmid expressing either WT β5 or β5Δpro) were streaked on FOA and grown at 30°C for 3 days (top) or 6 days.

Mutations of β6 and Ump1 have related effects on proteasome assembly. (A) The growth phenotype of β6Δ2-19. Strain MHY3040 was transformed with low-copy plasmids carrying the indicated alleles and then grown on FOA at 24°C to evaluate growth following eviction of the originally present URA3-marked WT β5 and β6 plasmids. (B) Suppression of β6Δ2-28 lethality by ump1Δ. Strains were grown on FOA at 24°C to evict the URA3/β6 plasmid originally present. (C) Native gel analysis of affinity-purified proteasomal particles from ump1 cells. Gel was stained with GelCode Blue. (D) Model for 20S assembly pathway showing new intermediates. Entry of Ump1, Pba1, and Pba2 proteins are shown but are not depicted in the complexes. Extensions on subunit ovals represent propeptides except for β7, where the extension depicts the C-terminal tail that inserts between the β1 and β2 subunits in the opposing β ring. The β5 propeptide, which is highlighted, is proposed to help align and join half-mers.