Fig. 5

Decreased activity of the RP₁CP species in proteasome-associated protein (PAP) mutants. pbac1-2, pbac3-1, ump1a-4, ump1b-1, pa200-2, cdc48b-1, cdc48c-2, cdc48d-2, and hsm3-1 mutants were grown for 10 days on half-strength MS media as shown in Fig. 4C. A Anti-PBA1, anti-PA200, and anti-HSP70 immunoblots of PAP mutant lysates resolved on 12% SDS-PAGE. B Quantitative analysis of Suc-LLVY-AMC (100 μM) stain signals of total lysates prepared and resolved on 4% Native-PAGE without any exogenous ATP. C, D Quantitative analysis of Suc-LLVY-AMC (100 μM) stain signals of total lysates prepared with 20 mM ATP and resolved on 4% Native-PAGE with 1 mM ATP. The native gels were stained and quantitated without (C) or with (D) incubation of 0.02% SDS to artificially activate the 20S CP. All mutant signals are expressed relative to that of Col-0, which is arbitrarily assigned a total signal value of 1.0. For gels run with exogenous ATP, quantifications of RP₂CP, RP₁CP, and free CP are expressed as a fraction of total sample signal within stacked bars. All bar charts display the means of three biological replicates ± SEM. Significant differences in RP₂CP, RP₁CP or free CP signal between each mutant and Col-0 were determined by independent Student’s t-tests (* = p < 0.05, ** = p < 0.01)