Figure 1. Human α4 can replace yeast α4 (Pre6) in yeast proteasomes and can form alternative ‘α4-α4’ proteasomes in mammalian cells.

(A) Human α4 (α4-Hs) expression in yeast allows growth in cells lacking Pre6 (α4-Sc) or both Pre6 and Pre9 (α3-Sc). Eviction of YCplac33-α4-Sc in α4Δ and α3Δ α4Δ yeast strains carrying either p424-GPD- α4-Hs or pRS424-α4-Sc results in viable colonies. The empty vector control showed no growth.

(B) Plate growth assay using serially diluted α4Δ (pre6Δ) and α3Δ α4Δ (pre9Δ pre96Δ) yeast cells transformed with either pRS424-PRE6 (α4-Sc) or p424GPD-PSMA7 (α4-Hs), which express yeast and human α4, respectively, and spotted on medium lacking tryptophan in 10-fold serial dilutions.

(C) Cartoon illustrating disulfide bond formation between engineered cysteines in α4 subunits if two α4 subunits are adjacent to each other in the α-ring.

(D) α4-Hs can form α4-α4 proteasomes in yeast. α4-Hs-CC (α4 T77C:T152C) subunits were disulfide crosslinked under oxidizing conditions (CuCl₂) in lysates, resolved by nonreducing SDS-PAGE, and immunoblotted with anti-α4. α4 monomer and dimer bands are indicated by black arrowheads. Additional uncharacterized oxidation products accumulated in both samples. Samples marked 4* and 8* were the same as those in lanes 4 and 8, respectively, but were treated with reducing agent (DTT). Also see Fig. S1A.

(E) Crosslinking of neighboring human α4 subunits can be detected in functional yeast 26S proteasomes purified (using native gel separation) from α3Δ α4Δ cells expressing α4-Hs-CC. No α4-α4 dimers are seen in the α4Δ single mutant, which still has endogenous α3 (lane 1), or with WT α4 that lacks the engineered cysteine residues (lane 2). Also see Fig. S1B.

(F) Ectopic expression of α4-Hs-CC results in formation of α4-α4 proteasomes in HEK293T cells. Whole cell extracts bearing disulfide-crosslinked α4 were analyzed as in panel D with yeast extracts except no CuCl₂ was added.