Stress-induced phospho-ubiquitin formation causes parkin degradation

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

In line with these findings, sub-cellular fractionation revealed similar amounts of phospho-poly- The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 Given the substantial presence of phospho-poly-Ub in the mitochondrial fraction after L-DOPA treatment and the affinity of parkin for phospho-Ub [47] , [72] , [73] , [81] , we predicted that we would observe mitochondrial parkin translocation following L-DOPA treatment, analogously to that observed following CCCP exposure [50] , [85] . In agreement with this, we saw a modest but significant increase of parkin in the mitochondrial fraction after 14-17 hours of L-DOPA treatment (1.31 ± 0.08 with L-DOPA vs. without, p = 0.01, N = 7) (Fig. 6A ,E), a time before L-DOPA-induced parkin loss is complete (Fig. 1B) . As a positive control, we examined mitochondrial translocation of parkin following 6 hours of CCCP exposure -a time at which parkin loss is underway but not complete (Fig. 10B ). Similarly to the effect of L-DOPA treatment, there was an increase of parkin in the mitochondrial fraction after CCCP treatment (1.55 ± 0.08 with CCCP vs. without, p = 0.007, N = 4) (Fig. 6B ,F). Altogether, these results suggest that parkin associates with stress-induced phospho-poly-Ub both in the cytosol and on mitochondria before its degradation.