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

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) following mitochondrial toxification in cell culture, alternative quality control pathways are suggested. To analyse 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, while pS65‐Ub accumulates in unstimulated parkin‐null flies, consistent with blocked degradation. Additionally, we show that pS65‐Ub specifically accumulates on disrupted mitochondria in vivo. 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 promoted turnover of both basal and paraquat‐induced pS65‐Ub in an Atg5‐null background. Thus, we have established that pS65‐Ub immunodetection can be used to analyse Pink1‐Parkin function in vivo as an alternative to reporter constructs. Moreover, our findings suggest that the Pink1‐Parkin pathway can promote mitochondrial turnover independently of canonical autophagy in vivo.