Parkin drives pS65-Ub turnover independently of canonical autophagy in Drosophila

Abstract Parkinson’s disease-related proteins, PINK1 and Parkin, act in a common pathway to maintain mitochondrial quality control. While the PINK1-Parkin pathway can promote autophagic mitochondrial turnover (mitophagy) in cell culture, recent studies have questioned whether they contribute to mitophagy in vivo, and alternative PINK1- and Parkin-dependent mitochondrial quality control pathways have been proposed. To determine the mechanisms by which the Pink1-Parkin pathway operates in vivo, we developed methods to detect Ser65-phosphorylated ubiquitin (pS65-Ub) in Drosophila. Exposure to the oxidant paraquat led to robust, Pink1-dependent pS65-Ub production. Surprisingly, parkin-null flies displayed strikingly elevated basal levels of pS65-Ub, suggestive of disrupted flux through the Pink1-parkin pathway. Depletion of the core autophagy proteins Atg1, Atg5 and Atg8a did not cause pS65-Ub accumulation to the same extent as loss of parkin, and overexpression of parkin was able to reduce both basal and paraquat-induced pS65-Ub levels in an Atg5-null background. Taken together, these results suggest that the Pink1-parkin pathway is able to promote mitochondrial turnover independently of canonical autophagy in vivo.