Fig. 5: Activity and inhibitor sensitivity of Arabidopsis ex-proteasomes.

Proteasome activity was assayed using the fluorogenic substrate Suc-LLVY-AMC. a Sensitivity of proteasomes in the CL and APF from Arabidopsis leaves to a collection of inhibitors designed to block either proteasomes (50 µM bortezomib (BTZ), 50 µM MG132, and 10 µM epoxomicin (Epo)), or various classes of peptidases/proteases (10 µM peptstatin, 15 µM bestatin, 100 µM PMSF, 10 µM leupeptin, and 10 µM E64), a 1X concentration of protease-inhibitor cocktail (PIC) that inhibits a collection of peptidases/proteases, or the activity of the CDC48 chaperone (10 µM CB-5083). The CL and APF were pretreated with the indicated concentrations for 5 min before assay. b Sensitivity of Suc-LLVY-AMC cleavage by the APF to a combination of 1X PIC and 50 µM BTZ. c and d, Association of Suc-LLVY-AMC cleavage with ex-proteasomes as judged by immune-depletion assays using a transgenic Arabidopsis line where the CP α7 subunit PAG1 was replaced with a FLAG-tagged variant. A resuspended APFp from PAG1-FLAG pag1-1 leaves was depleted of proteasomes by immunoprecipitation with anti-FLAG beads. The APFp and the immunoprecipitated (IP) and supernatant (Sup) fractions were assayed for (c) protease activity using Suc-LLVY-AMC or for (d) proteasome subunits by immunoblotting with antibodies against CP (PAG1 and PBA1) and RP (RPN1) subunits or the FLAG epitope. Bars in panels a-c reflect the mean (±SD) of three technical replicates. Different letters above the bars indicate a significant difference from others using a one-way ANOVA followed by Tukey’s test to determine significance. Individual data points are included.