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. 2015 Mar 23;112(14):E1763–E1772. doi: 10.1073/pnas.1423319112

Fig. 3.

Fig. 3.

Rv3780 is an ATP-independent proteasome activator. (A) Rv3780 stimulates proteasomal degradation of a tetrapeptide substrate. Recombinant 20S CP–His6 and His6–Rv3780 were purified from E. coli, mixed, and incubated with Suc-LLVY-AMC. Data are shown as fold change in degradation rate compared with the 20S CP–His6 alone. Molar ratios represent His6–Rv3780 rings:20S CPs. (Inset) Absolute degradation rate in relative fluorescence units (RFU) generated per minute for the 20S CP–His6 alone or with a 58.3-fold molar excess of His6–Rv3780. (B) Rv3780 stimulates proteasomal degradation of a nonapeptide substrate. Reactions were performed as in A, except with LF-2 used as a substrate. Rv3780Y173A, mutant Rv3780 that was unable to bind 20S CPs (Fig. 1C). (C) Rv3780 enhances proteasomal degradation of an unfolded protein. Recombinant purified 20S CP–His6 and His6–Rv3780 were mixed with FITC-labeled β-casein at room temperature. Samples were removed at the indicated time points and separated by 12% (wt/vol) SDS/PAGE, and FITC–β-casein was visualized and quantified by in-gel fluorescence.