Stress-induced phospho-ubiquitin formation causes parkin degradation

Lyudmila Kovalchuke; Eugene V. Mosharov; Oren A. Levy; Lloyd A. Greene

For most experiments, values were normalized to the control condition in that experiment before replicate experiments were combined. Paired t-tests were performed in most cases, . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/484857 doi: bioRxiv preprint followed by Holm correction for multiple comparisons. In time-course analyses, unpaired t-tests at each time point followed by Holm-Sidak's multiple comparisons test were used. For analysis of differences between parkin mutants, one-way ANOVA of stressor-treated mutants was performed, followed by Holm-Sidak correction for multiple comparisons. Differentiated PC12 cells were transduced for 3 to 6 days with lentiviral vectors carrying the indicated parkin mutants and bicistronically expressed GFP before cells were harvested for Western immunoblotting. Representative blots are shown. To compare relative expression levels of the different parkin mutants, parkin levels for each construct were normalized first to ERK1 and 2 to control for loading differences and then to GFP to control for variability in viral titer. The results of this quantification are shown on the right. Error bars show SEM from 5-10 independent experiments; * p ≤ 0.05 relative to WT by paired t-test with Holm correction for multiple comparisons. Figure 5 . Association with phospho-Ub leads to parkin loss from CCCP treatment A,B. Differentiated PC12 cells were transduced with lentiviral vectors carrying the indicated parkin mutants. Three to five days after infection, cells were treated with 10 μM CCCP for 6 (A) or 12 (B) hours before harvest for Western immunoblotting and assessment of parkin protein levels. Representative immunoblots and quantifications of parkin levels are shown. The level of each parkin mutant after CCCP treatment was normalized to the level of the same mutant after control treatment, which was set to 1 in each case. The latter is represented by the leftmost gray bar in the quantifications. Error bars show SEM from N = 4-6 (A) and 3-4 (B) independent experiments; *** p ≤ 0.001, **** p ≤ 0.0001 relative to WT by one-way ANOVA of CCCP-treated mutants with Holm-Sidak's multiple comparisons test. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/484857 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/484857 doi: bioRxiv preprint Figure 10 . Mitophagy is not required for phospho-Ub-dependent parkin degradation A,B. L-DOPA and CCCP do not induce changes in mitochondrial protein levels consistent with robust mitophagy. Differentiated PC12 cells were treated with 200 μM L-DOPA (A) or 10 μM CCCP (B) for the indicated times before harvest for WB. Representative blots and quantifications of the indicated proteins from N = 3 independent experiments are shown. A. L-DOPA does not significantly decrease levels of UQCRC1, VDAC, Tim23, or Tom20, while it does induce loss of parkin and Opa1-long. B. CCCP does not significantly decrease levels of UQCRC1, VDAC, or Tom20, while it does induce loss of parkin, Tim23, and Opa1-long. Error bars show SEM; * p ≤ 0.05 by unpaired ttest, with Holm-Sidak correction for multiple comparisons. Significance was queried between untreated and drug-treated groups at each time point. The copyri