Stress-induced phospho-ubiquitin formation causes parkin degradation

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

Given that parkin was clearly active in PC12 cells after CCCP treatment (Fig. 8A,B; Fig. 9A ,B), we also sought to determine whether this activity led to mitophagy in CCCP-treated PC12 cells. Examining the same proteins as we did for L-DOPA treatment, we did not observe ubiquitination or loss consistent with mitophagy in cells exposed to CCCP (Fig. 10B) . Levels of Tim23 did decrease with CCCP (15.1 ± 2.6% decrease after 5 hours relative to time zero, p = 0.01 vs. no treatment, N = 3), but those of VDAC, Tom20, and UQCRC1 remained unchanged ( Fig. 10B ). As with L-DOPA treatment, parkin loss was still robust in these experiments (47.6 ± 4.3% loss after 25 hours, p = 0.002 vs. no treatment, N = 3) (Fig. 10B) , and levels of the long isoform of OPA1 dropped precipitously in response to the mitochondrial membrane potential loss (83.5 ± 7.1% decrease after 25 hours relative to time zero, p = 0.001 vs. no treatment, N = 3) (Fig. 10B) . These results indicate that even when parkin is clearly active and Mfn2 has been poly-ubiquitinated, mitophagy may not necessarily take place, consistent with a previous report . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 [40] . Altogether, our data indicate that phospho-Ub-mediated parkin degradation does not require canonical mitochondrial parkin activity or mitophagy.