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, and gRNAs sequences are in Table S1. b. The experimental setup used in this study. Cells were pulse treated with Btz or Cfz for 1h, then cultured in drug-free media for times indicated and analyzed as described. c. The viability of wt- and DDI2 KO clones of HAP1 cells was measured using CellTiter-Glo, and the inhibition of β5 sites was measured with the Proteasome-Glo assay at times indicated; n=2-5. d. Knockout of DDI2 inhibits the Nrf1 processing. Western blots of Btz-treated HAP1 cells. The sample in the first lane is wt cells treated with VCP/p97 inhibitor CB-5083 immediately after removal of Btz. VCP inhibitors blocks Nrf1 processing [17–19]. The full-size membranes and proteasome activity measurements from this experiment are presented in Fig. S1b and S1c. e. MDA-MB-231 and SUM149 cells were analyzed by western blot 72h after transfection with DDI2 siRNAs (Table S3). The full-size membranes are presented in Fig. S3. f. The β5 activity in siRNA-transfected SUM149 and MDA-MB-231 was measured using Suc-LLVY-AMC immediately and 18h after treatment with 100 nM Btz; n=3. g. β5 activity was measured in HCT-116 cells with the Proteasome-Glo assay immediately and 18h after treatment with PIs; n=2.

Proteasome activity recovers before upregulation of proteasome gene expression.

Wt-HAP1 cells were pulse-treated with Btz (100 nM), cultured in a 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; n=2-5. b. In a parallel experiment, the mRNA was isolated, and the expression of proteasome genes was quantified using quantitative RT-PCR; n=3. Results of the t-test at h are in parenthesis.

The recovery of proteasome activity requires protein synthesis.

a. Wt-HAP1 and DDI2 KO cells were treated for 1h at indicated concentrations of Btz and Cfz and then cultured in a drug-free media in the absence (solid lines) or presence (dashed lines) of CHX. 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; n=3-4. 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 fractions as a % of total is shown; n=2.

Escape from rapid degradation of nascent subunits can explain rapid recovery of proteasome activity.

a. Turnover of proteasome subunit in human RPE-1 cells was measured by quantitative mass-spectrometry following 1h labeling with heavy isotopes. Data from Table S4 in [44]. b. Proposed model of how nascent proteasome subunits are partitioned between assembly and degradation.