Recovery of proteasome activity is DDI2 independent.

a. Expression of DDI2 in the CRISPR-generated clones of HAP1 cells used in this work was analyzed by western blot. The full-size membrane is presented in Fig. S1a. b. The experimental set up in this study. Cells were pulse treated with Btz and Cfz for 1hr, then cultured in a drug-free media, and analyzed at times indicated. c. Wt- and DDI2 KO clones of HAP1 cells were treated for 1hr. The cell viability was measured using CellTiter-Glo, and the inhibition of β5 sites was measured with the Proteasome-Glo assay at times indicated. Data are averages+/-S.E.M. of two to five biological replicates d. Knockout of DDI2 inhibits the Nrf1 processing. Western blots of Btz-treated HAP1 cells. Sample in the first lane are wt cells treated fwith VCP/p97 inhibitor CB-5083, which blocks Nrf1 processing [17-19], immediately after removal of Btz. The full-size membranes and proteasome activity measurements from this experiment are presented in Figs. S1b and S1c. e. MDA-MB-231 and SUM149 cells were analyzed by western blot 72hr after transfection with DDI2 siRNAs. The full-size membranes are presented in Fig. S2. f. The β5 activity in siRNA-transfected SUM149 and MDA-MB-231 treated with 100 nM Btz for 1hr. The β5 activity was measured using Suc-LLVY-AMC immediately and 18 hrs after treatment. Data are averages+/-S.E.M. of three to five biological replicates. Statistical analysis was conducted using mixed-effect multiple comparisons; p-values ≤ 0.05 were considered significant.

Proteasome activity recovers before up-regulation of proteasome gene expression.

Wt-HAP1 cells were pulse-treated with Btz (100 nM), cultured in drug-free medium, and analyzed at indicated times. a. β5 activity was measured using Proteasome-Glo and normalized first to CellTiter-Glo viability data, and then to proteasome activity in the mock-treated samples. The data are averages±S.E.M of two to five biological replicates. b. In a parallel experiment, the mRNA was isolated, and the expression of proteasome genes was quantified using quantitative RT-PCR. Data are averages±S.E.M. of three biological replicates. The number in parenthesis indicates the t-test results at an 8hr time point.

The recovery of proteasome activity required the synthesis of new proteasomes.

a. Wt (blue) and DDI2 KO (red) HAP1 cells were treated for 1hr at indicated concentrations of Btz and Cfz and then cultured in a drug-free media in the absence (solid line) or presencence of CHX (dashed line). The β5 activity was measured using Proteasome-Glo and normalized first to cell viability which was determined in a parallel experiment using CellTiter-Glo and then to untreated controls. Data are averages+/-S.E.M of two to five biological replicates. b. All proteasome mRNAs are actively translated. mRNA isolated from untreated wt-HAP1 cells were analyzed by polysome profiling. The combined % mRNAs in the 80S and polysomal fraction relative to the cumulative amount of mRNA in all fractions are shown. Data are averages+/-S.E.M of two biological replicates.

Rapid degradatio of nascent proteasome polypeptides can explain the rapid recovery of activity.

a. Turnover of proteasome subunit in human RPE cells was measured by quantitative mass-spectrometry following 1hr labeling with heavy isotopes. Data from Table S4 in [23]. b. Proposed model of how nascent proteasome subunits are partitioned between assembly and degradation.