Jcb_201402104 1..11

Loss-of-function mutations in PINK1 and Parkin cause early onset Parkinson’s disease (Kitada et al., 1998; Valente et al., 2004). Genetic studies in Drosophila and cell biology studies in mammalian cells place PINK1 upstream of Parkin in the same pathway and indicate they may normally mediate mitochondrial quality control (Narendra et al., 2012; Ashrafi and Schwarz, 2013; Winklhofer, 2014). PINK1 is a kinase that is imported into mitochondria, cleaved by the inner membrane protease PARL to generate an N-end degron and then eliminated by the proteasome (Lin and Kang, 2008; Jin et al., 2010; Deas et al., 2011; Yamano and Youle, 2013). When mitochondria lose membrane potential or amass unfolded protein, PINK1 accumulates on the outer membrane via TOM7 in association with the TOM complex (Hasson et al., 2013). On the outer mitochondrial membrane (OMM), PINK1 recruits the E3 ubiquitin ligase Parkin (Geisler et al., 2010; Narendra et al., 2010; Vives-Bauza et al., 2010) and activates latent Parkin activity (Matsuda et al., 2010) to ubiquitinate scores of OMM proteins (Sarraf et al., 2013). This leads to proteasomal degradation of OMM proteins (Tanaka et al., 2010; Chan et al., 2011; Yoshii et al., 2011) and to selective autophagy of damaged mitochondria (Narendra et al., 2008), suggesting that PINK1 and Parkin mediate a mitochondrial quality control pathway. How PINK1 recruits Parkin to the OMM and what PINK1 kinase substrate is involved have been unclear. A leading candidate is Parkin itself, as PINK1 directly phosphorylates Parkin at serine 65 (S65; Kondapalli et al., 2012; Shiba-Fukushima et al., 2012). This model is consistent with data that PINK1 experimentally localized to peroxisomes or lysosomes can recruit Parkin to these locations (Lazarou et al., 2012). Here we show that PINK1 recruits Parkin to mitochondria despite mutation of S65 to alanine or individual mutation of all other Ser/Thr residues conserved between Drosophila and human Parkin. This indicates that another PINK1 substrate mediates Parkin translocation and activation. Using mass spectrometry we identified ubiquitin (Ub) as an endogenous PINK1 substrate and found that both a phosphomimetic mutant Ub in cells and phospho-Ub in vitro can activate Parkin. Interestingly, PINK1 PINK1 kinase activates the E3 ubiquitin ligase Parkin to induce selective autophagy of damaged mitochondria. However, it has been unclear how PINK1 activates and recruits Parkin to mitochondria. Although PINK1 phosphorylates Parkin, other PINK1 substrates appear to activate Parkin, as the mutation of all serine and threonine residues conserved between Drosophila and human, including Parkin S65, did not wholly impair Parkin translocation to mitochondria. Using mass spectrometry, we discovered that endogenous PINK1 phosphorylated ubiquitin at serine 65, homologous to the site phosphorylated by PINK1 in Parkin’s ubiquitin-like domain. Recombinant TcPINK1 directly phosphorylated ubiquitin and phosphoubiquitin activated Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin. Furthermore, expression of ubiquitin S65A, a mutant that cannot be phosphorylated by PINK1, inhibited Parkin translocation to damaged mitochondria. These results explain a feedforward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity. PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity