Figure 4: In vitro reconstitution of the S. acidocaldarius 20S proteasome.
(a) Ni-NTA agarose IMAC chromatography purification step following coexpression of the untagged Saci0613 α-subunit with the Saci0662 (His-tagged) and Saci0909 β-subunits. Lane 1: Saci0613 plus Saci0662; lane 2: Saci0613 plus Saci0909, lane 3: Saci0613 plus Saci0662ΔN, lane 4: Saci0613 plus Saci0909ΔN; lane 5: Saci0613 plus Saci0662ΔN and Saci0909ΔN. The truncated regions in Saci0662ΔN and Saci0909ΔN, respectively, are indicated by vertical arrows in the schematic below. (b) Size exclusion chromatography (SEC) analysis of the Saci0613/Saci0662ΔN/Saci0909ΔN catalytically active, and Saci0613/Saci0662ΔN inactive 20S proteasome complex. Chromatography UV traces are displayed for both elution profiles. Both peaks are consistent with a complex of ≈660 kDa. (c) Transmission electron micrographs (TEM) of the Saci0613/Saci0662ΔN/Saci0909ΔN 20S complex (1 and 2), and the Saci0613/Saci0662ΔN inactive 20S complex (3 and 4), negatively stained with uranyl acetate. The left hand panels are at 50 000X magnification and the right hand panels at × 100 000 magnification; 100 or 20 nm scale bars are shown, respectively. (d) Free Urm1 is processed by the active 20S proteasome complex, and degradation is stimulated by the addition of the PAN regulatory ATPase. 30 μg of Urm1 protein was incubated with 20 μg of the proteasome, with or without 20 μg of the PAN ATPase complex for 1 h at 70 °C; all reactions contained 5 mM ATP. Lane 1: Urm1 control with the inactive proteasome; lane 2: Urm1 degradation by the active proteasome; lane 3: Urm1 degradation by the active proteasome stimulated by the PAN ATPase. (e) SEC-MALS analysis of the Saci0613/Saci0662ΔN/Saci0909ΔN catalytically active 20S proteasome. The complex has a fitted molecular weight of 658.1 kDa (±0.166%), consistent with a 28-subunit cylindrical proteasome assembly, with a polydispersity of 1.000 (±0.234%). Differential refractive index (dRI) and light scattering (LS) are plotted in conjunction with molecular weight (Mw).
