No abstract

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.

Dysfunction of PINK1, a mitochondrial Ser/Thr kinase, causes familial Parkinson's disease (PD). Recent studies have revealed that PINK1 is rapidly degraded in healthy mitochondria but accumulates on the membrane potential (ΔΨm)-deficient mitochondria, where it recruits another familial PD gene product, Parkin, to ubiquitylate the damaged mitochondria. Despite extensive study, the mechanism underlying the homeostatic control of PINK1 remains unknown. Here we report that PINK1 is autophosphorylated following a decrease in ΔΨm and that most disease-relevant mutations hinder this event. Mass spectrometric and mutational analyses demonstrate that PINK1 autophosphorylation occurs at Ser228 and Ser402, residues that are structurally clustered together. Importantly, Ala mutation of these sites abolishes autophosphorylation of PINK1 and inhibits Parkin recruitment onto depolarized mitochondria, whereas Asp (phosphorylation-mimic) mutation promotes mitochondrial localization of Parkin even though autophosphorylation was still compromised. We propose that autophosphorylation of Ser228 and Ser402 in PINK1 is essential for efficient mitochondrial localization of Parkin.

Mutations in CHCHD2 have been identified in some Parkinson's disease (PD) cases. To understand the physiological and pathological roles of CHCHD2, we manipulated the expression of CHCHD2 in Drosophila and mammalian cells. The loss of CHCHD2 in Drosophila causes abnormal matrix structures and impaired oxygen respiration in mitochondria, leading to oxidative stress, dopaminergic neuron loss and motor dysfunction with age. These PD-associated phenotypes are rescued by the overexpression of the translation inhibitor 4E-BP and by the introduction of human CHCHD2 but not its PD-associated mutants. CHCHD2 is upregulated by various mitochondrial stresses, including the destabilization of mitochondrial genomes and unfolded protein stress, in Drosophila. CHCHD2 binds to cytochrome c along with a member of the Bax inhibitor-1 superfamily, MICS1, and modulated cell death signalling, suggesting that CHCHD2 dynamically regulates the functions of cytochrome c in both oxidative phosphorylation and cell death in response to mitochondrial stress.

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.