Figure 6. Backside mutants impair K48 chain debranching and UCH37-dependent proteasomal degradation.

(A) Steady-state kinetic analyses of the cleavage of K6/K48 tri-ubiquitin (Ub) with different UCH37 variants (WT, F117A, F121A, and I216E) in complex with RPN13. The data corresponding to WT and I216E were fit to the nonlinear Michaelis-Menten equation. The linear equation, rate = k_cat/K_m[K6/K48 tri-Ub][Enzyme], was sufficient to fit the data corresponding to L181A, F117A, and F121A. (B) SDS-PAGE analysis of the cleavage of K6/K48 and K48/K63 high-molecular weight (HMW) Ub chains with different UCH37 variants (WT, F117A, F121A, and I216E) in complex with RPN13. Gels were imaged by Sypro Ruby staining. (C) Western blot analysis of cleavage of K6/K48 HMW Ub chains by WT Ptsm, WT UCH37•RPN13-replenished Ptsm (rWT), UCH37^F117A•RPN13-replenished Ptsms (rF117A), UCH37^F121A•RPN13-replenished Ptsms (rF121A), and UCH37^I216E•RPN13-replenished Ptsms (rI216E). 0.2 mg of Ptsm complex was used in each reaction. Immunoblotting was performed with Alexa Fluor 647-conjugated streptavidin. (D) Fluorescent SDS-PAGE analyses of the degradation of Cy5-labeled, K48/K63-Ub_n-titin-I27^V15P-23-K-35 and K11/K48-polyUb-UBE2S-UBD using WT, rWT, rC88A, rI216E, rF117A, and rF121A Ptsm complexes. Reactions were performed under multiturnover conditions and quenched after 1 hr. Gels stained for total protein are shown for reference. (E) Western blot analysis of UCH37 levels in WT, UCH37^KO (KO), and KO HEK293 cells expressing UCH37 variants under a doxycycline-inducible promoter. Immunoblotting was performed with α-UCH37 and α-actin antibodies. (F) Schematic showing the different degradation profiles of the Ptsm activity reporter GFP^u in WT and UCH37^KO HEK293 cells expressing UCH37 variants under a doxycycline-inducible promoter. (G) Plots showing percent GFP^u remaining in the indicated cell lines after shutting off translation with emetine. (H) Scheme showing the labeling and visualization of newly synthesized proteins (NSPs) with azidohomoalanine (AHA) in UCH37^KO cells expressing either UCH37^I216E or UCH37^F117A. (I) Fluorescent SDS-PAGE analysis of the turnover of AHA-labeled NSPs in UCH37^KO cells expressing either UCH37^I216E or UCH37^F117A. (J) Quantitation of the results shown in (I).

Figure 6—figure supplement 1. Backside mutants impair K48 chain debranching and UCH37-dependent proteasomal degradation.

(A) Isothermal titration calorimetry analyses of mono-ubiquitin (Ub) and K48 di-Ub binding to UCH37^C88A/I216E•RPN13. (B–H) Western blot analyses of the purification of different 26 S Ptsm complexes with and without UCH37 variants. (I) Hydrolysis of Ub-AMC by the indicated 26 S Ptsm complexes. (J) Hydrolysis of suc-LLVY-AMC by the indicated 26 S Ptsm complexes.