Parkinson's disease genes PINK1 and parkin encode kinase and ubiquitin ligase, respectively. The gene products PINK1 and Parkin are implicated in mitochondrial autophagy, or mitophagy. Upon the loss of mitochondrial membrane potential (ΔΨm), cytosolic Parkin is recruited to the mitochondria by PINK1 through an uncharacterised mechanism – an initial step triggering sequential events in mitophagy. This study reports that Ser65 in the ubiquitin-like domain (Ubl) of Parkin is phosphorylated in a PINK1-dependent manner upon depolarisation of ΔΨm. The introduction of mutations at Ser65 suggests that phosphorylation of Ser65 is required not only for the efficient translocation of Parkin, but also for the degradation of mitochondrial proteins in mitophagy. Phosphorylation analysis of Parkin pathogenic mutants also suggests Ser65 phosphorylation is not sufficient for Parkin translocation. Our study partly uncovers the molecular mechanism underlying the PINK1-dependent mitochondrial translocation and activation of Parkin as an initial step of mitophagy.

The kinase PINK1 and the E3 ubiquitin (Ub) ligase Parkin participate in mitochondrial quality control. The phosphorylation of Ser65 in Parkin's ubiquitin-like (UBl) domain by PINK1 stimulates Parkin activation and translocation to damaged mitochondria, which induces mitophagy generating polyUb chain. However, Parkin Ser65 phosphorylation is insufficient for Parkin mitochondrial translocation. Here we report that Ser65 in polyUb chain is also phosphorylated by PINK1, and that phosphorylated polyUb chain on mitochondria tethers Parkin at mitochondria. The expression of Tom70MTS-4xUb SE, which mimics phospho-Ser65 polyUb chains on the mitochondria, activated Parkin E3 activity and its mitochondrial translocation. An E3-dead form of Parkin translocated to mitochondria with reduced membrane potential in the presence of Tom70MTS-4xUb SE, whereas non-phospho-polyUb mutant Tom70MTS-4xUb SA abrogated Parkin translocation. Parkin binds to the phospho-polyUb chain through its RING1-In-Between-RING (IBR) domains, but its RING0-linker is also required for mitochondrial translocation. Moreover, the expression of Tom70MTS-4xUb SE improved mitochondrial degeneration in PINK1-deficient, but not Parkin-deficient, Drosophila. Our study suggests that the phosphorylation of mitochondrial polyUb by PINK1 is implicated in both Parkin activation and mitochondrial translocation, predicting a chain reaction mechanism of mitochondrial phospho-polyUb production by which rapid translocation of Parkin is achieved.

Understanding how lipid dynamics change with membrane curvature is important given that biological membranes constantly change their curvature and morphology through membrane fusion and endo-/exocytosis. Here, we used time-resolved small-angle neutron scattering and time-resolved fluorescence to characterize the properties and dynamics of phospholipids in vesicles with different curvatures. Dissociation of phospholipids from vesicles required traversing an energy barrier comprising positive enthalpy and negative entropy. However, lipids in membranes with high positive curvature have dense acyl chain packing and loose headgroup packing, leading to hydrophobic hydration due to water penetration into the membrane. These properties were found to lower the hydrophobic hydration enhancement associated with phospholipid dissociation and mitigate the acyl chain packing of lipids adjacent to the space created by the lipid dissociation, resulting in an increase in activation entropy. The results of this study provide important insights into the functions of biomembranes in relation to their dynamic structural changes.

The mitochondrial kinase PTEN-induced kinase 1 (PINK1) and cytosolic ubiquitin ligase (E3) Parkin/PRKN are involved in mitochondrial quality control responses. PINK1 phosphorylates ubiquitin and the Parkin ubiquitin-like (Ubl) domain at serine 65 and promotes Parkin activation and translocation to damaged mitochondria. Upon Parkin activation, the Ubl domain is ubiquitinated at lysine (K) 27 and K48 residues. However, contribution of K27/K48 ubiquitination towards Parkin activity remains unclear. In this study, ubiquitination of K56 (corresponding to K27 in the human), K77 (K48 in the human), or both, was blocked by generating Drosophila Parkin (dParkin) mutants to examine the effects of Parkin Ubl domain ubiquitination on Parkin activation in Drosophila. The dParkin, in which K56 was replaced with arginine (dParkin K56R), rescued pupal lethality in flies by co-expression with PINK1, whereas dParkin K77R could not. The dParkin K56R exhibited reduced abilities of mitochondrial fragmentation and motility arrest, which are mediated by degrading Parkin E3 substrates Mitofusin and Miro, respectively. Pathogenic dParkin K56N, unlike dParkin K56R, destabilized the protein, suggesting that not only was dParkin K56N non-ubiquitin-modified at K56 but also the structure of the Ubl domain for activation was largely affected. Ubiquitin attached to K27 of the Ubl domain during PINK1-mediated Parkin activation was likely to be phosphorylated because human Parkin K27R weakened Parkin self-binding and activation in trans. Therefore, our findings suggest a new mechanism of Parkin activation, where an activation complex is formed through phospho-ubiquitin attachment on the K27 residue of the Parkin Ubl domain.