Phosphorylation is often used to promote protein ubiquitylation, yet we rarely understand quantitatively how ligase activation and ubiquitin (UB) chain assembly are integrated with phospho-regulation. Here we employ quantitative proteomics and live-cell imaging to dissect individual steps in the PINK1 kinase-PARKIN UB ligase mitochondrial control pathway disrupted in Parkinson’s Disease. PINK1 plays a dual role by phosphorylating PARKIN on its UB-like domain and poly-UB chains on mitochondria. PARKIN activation by PINK1 produces canonical and non-canonical UB chains on mitochondria, and PARKIN-dependent chain assembly is required for accumulation of poly-phospho-UB (poly-p-UB) on mitochondria. In vitro, PINK1 directly activates PARKIN’s ability to assemble canonical and non-canonical UB chains, and promotes association of PARKIN with both p-UB and poly-p-UB. Our data reveal a feed-forward mechanism that explains how PINK1 phosphorylation of both PARKIN and poly-UB chains synthesized by PARKIN drives a program of PARKIN recruitment and mitochondrial ubiquitylation in response to mitochondrial damage.

Damaged mitochondria are detrimental to cellular homeostasis. One mechanism for removal of damaged mitochondria involves the PINK1-PARKIN pathway, which poly-ubiquitylates damaged mitochondria to promote mitophagy. We report that assembly of ubiquitin chains on mitochondria triggers the recruitment of autophagy adaptors concomitantly with activation of the TBK1 protein kinase, which physically associates with OPTN, NDP52, and SQSTM1. Full TBK1 activation in HeLa cells requires OPTN and NDP52, and OPTN ubiquitin chain binding. In addition to the known role of S177 phosphorylation in OPTN on ATG8 recruitment, TBK1-dependent phosphorylation on S473 and S513 promotes ubiquitin chain binding in vitro as well as TBK1 activation, OPTN mitochondrial retention, and efficient mitophagy in vivo. These data reveal a self-reinforcing positive feedback mechanism that coordinates TBK1-dependent autophagy adaptor phosphorylation with the assembly of ubiquitin chains on mitochondria to facilitate efficient mitophagy. Analogous mechanisms may facilitate adaptor-protein mediated delivery of other types of cargo to autophagosomes.

Mitochondria produce energy in the form of ATP via oxidative phosphorylation. As defects in oxidative phosphorylation can generate harmful reactive oxygen species, it is important that damaged mitochondria are efficiently removed via a selective form of autophagy known as mitophagy. Owing to a combination of cell biological, structural and proteomic approaches, we are beginning to understand the mechanisms by which ubiquitin-dependent signals mark damaged mitochondria for mitophagy. This Review discusses the biochemical steps and regulatory mechanisms that promote the conjugation of ubiquitin to damaged mitochondria via the PTEN-induced putative kinase 1 (PINK1) and the E3 ubiquitin-protein ligase parkin and how ubiquitin chains promote autophagosomal capture. Recently discovered roles for parkin and PINK1 in the suppression of mitochondrial antigen presentation provide alternative models for how this pathway promotes the survival of neurons. A deeper understanding of these processes has major implications for neurodegenerative diseases, including Parkinson disease, where defects in mitophagy and other forms of selective autophagy are prominent.

The PTEN-induced putative kinase protein 1 (PINK1) and ubiquitin (UB) ligase PARKIN direct damaged mitochondria for mitophagy. PINK1 promotes PARKIN recruitment to the mitochondrial outer membrane (MOM) for ubiquitylation of MOM proteins with canonical and noncanonical UB chains. PINK1 phosphorylates both Ser65 (S65) in the UB-like domain of PARKIN and the conserved Ser in UB itself, but the temporal sequence and relative importance of these events during PARKIN activation and mitochondria quality control remain poorly understood. Using "UB S65A-replacement," we find that PARKIN phosphorylation and activation, and ubiquitylation of Lys residues on a cohort of MOM proteins, occur similarly irrespective of the ability of the UB-replacement to be phosphorylated on S65. In contrast, polyubiquitin (poly-UB) chain synthesis, PARKIN retention on the MOM, and mitophagy are reduced in UB S65A -replacement cells. Analogous experiments examining roles of individual UB chain linkage types revealed the importance of K6 and K63 chain linkages in mitophagy, but phosphorylation of K63 chains by PINK1 did not enhance binding to candidate mitophagy receptors optineurin (OPTN), sequestosome-1 (p62), and nuclear dot protein 52 (NDP52) in vitro. Parallel reaction monitoring proteomics of total mitochondria revealed the absence of p-S65-UB when PARKIN cannot build UB chains, and <0.16% of the monomeric UB pool underwent S65 phosphorylation upon mitochondrial damage. Combining p-S65-UB and p-S65-PARKIN in vitro showed accelerated transfer of nonphosphorylated UB to PARKIN itself, its substrate mitochondrial Rho GTPase (MIRO), and UB. Our data further define a feed-forward mitochondrial ubiquitylation pathway involving PARKIN activation upon phosphorylation, UB chain synthesis on the MOM, UB chain phosphorylation, and further PARKIN recruitment and enzymatic amplification via binding to phosphorylated UB chains.he RING-Between-RING ubiquitin (UB) ligase PARKIN and the PTEN-induced putative kinase protein 1 (PINK1), both of which are mutated in Parkinson's disease, promote ubiquitylation of numerous outer membrane proteins on damaged mitochondria, which facilitates mitophagy (1). When mitochondria are healthy, cytoplasmic PARKIN is thought to be in an unphosphorylated and auto-inhibited state (2-6). Upon mitochondrial damage, PINK1 is stabilized on the mitochondrial outer membrane (MOM) and promotes phosphorylation of both Ser65 (S65) on PARKIN's UB-like (UBL) domain and the conserved S65 residue in UB itself, which is 62% similar to PARKIN's UBL (1,(7)(8)(9)(10)(11)(12)(13)(14). PARKIN is also retained on the mitochondrial surface and ubiquitylates numerous MOM proteins through the formation of both canonical and noncanonical UB chains. The order of events, as well as the precise roles of UB and PARKIN phosphorylation, is poorly defined, and multiple models have been proposed. All models agree that PARKIN activation reverses auto-inhibition but differ in the mechanism and role of p-S65-UB (8,(11)(12)(13)(14)(15)). In one model, binding...

Flux through kinase and ubiquitin-driven signaling systems depends on the modification kinetics, stoichiometry, primary site specificity, and target abundance within the pathway, yet we rarely understand these parameters and their spatial organization within cells. Here we develop temporal digital snapshots of ubiquitin signaling on the mitochondrial outer membrane in embryonic stem cell-derived neurons, and we model HeLa cell systems upon activation of the PINK1 kinase and PARKIN ubiquitin ligase by proteomic counting of ubiquitylation and phosphorylation events. We define the kinetics and site specificity of PARKIN-dependent target ubiquitylation, and we demonstrate the power of this approach to quantify pathway modulators and to mechanistically define the role of PARKIN UBL phosphorylation in pathway activation in induced neurons. Finally, through modulation of pS65-Ub on mitochondria, we demonstrate that Ub hyper-phosphorylation is inhibitory to mitophagy receptor recruitment, indicating that pS65-Ub stoichiometry in vivo is optimized to coordinate PARKIN recruitment via pS65-Ub and mitophagy receptors via unphosphorylated chains.

TBK1 phosphorylates RAB7A-S72 to support multiple downstream steps in PARKIN-dependent mitophagy.

The PINK1 protein kinase activates the PARK2 ubiquitin ligase to promote mitochondrial ubiquitylation and recruitment of ubiquitin-binding mitophagy receptors typified by OPTN and TAX1BP1. Here, we combine proximity biotinylation of OPTN and TAX1BP1 with CRISPR-Cas9–based screens for mitophagic flux to develop a spatial proteogenetic map of PARK2-dependent mitophagy. Proximity labeling of OPTN allowed visualization of a “mitochondrial-autophagosome synapse” upon mitochondrial depolarization. Proximity proteomics of OPTN and TAX1BP1 revealed numerous proteins at the synapse, including both PARK2 substrates and autophagy components. Parallel mitophagic flux screens identified proteins with roles in autophagy, vesicle formation and fusion, as well as PARK2 targets, many of which were also identified via proximity proteomics. One protein identified in both approaches, HK2, promotes assembly of a high–molecular weight complex of PINK1 and phosphorylation of ubiquitin in response to mitochondrial damage. This work provides a resource for understanding the spatial and molecular landscape of PARK2-dependent mitophagy.

Mitophagy, the selective recycling of mitochondria through autophagy, is a crucial metabolic process induced by cellular stress, and defects are linked to aging, sarcopenia, and neurodegenerative diseases. To therapeutically target mitophagy, the fundamental in vivo dynamics and molecular mechanisms must be fully understood. Here, we generated mitophagy biosensor zebrafish lines expressing mitochondrially targeted, pH-sensitive, fluorescent probes mito-Keima and mito-EGFP-mCherry and used quantitative intravital imaging to illuminate mitophagy during physiological stresses—embryonic development, fasting and hypoxia. In fasted muscle, volumetric mitolysosome size analyses documented organelle stress-response dynamics, and time-lapse imaging revealed mitochondrial filaments undergo piecemeal fragmentation and recycling rather than the wholesale turnover observed in cultured cells. Hypoxia-inducible factor (Hif) pathway activation through physiological hypoxia or chemical or genetic modulation also provoked mitophagy. Intriguingly, mutation of a single mitophagy receptor bnip3 prevented this effect, whereas disruption of other putative hypoxia-associated mitophagy genes bnip3la (nix), fundc1, pink1 or prkn (Parkin) had no effect. This in vivo imaging study establishes fundamental dynamics of fasting-induced mitophagy and identifies bnip3 as the master regulator of Hif-induced mitophagy in vertebrate muscle.