Selective localization of Mfn2 near PINK1 enable its preferential ubiquitination by Parkin on mitochondria

Mutations in Parkin and PINK1 cause an early-onset familial Parkinson’s disease. Parkin is a RING-In-Between-RING (RBR) E3 ligase that transfers ubiquitin from an E2 enzyme to a substrate in two steps: 1) thioester intermediate formation on Parkin, and 2) acyl transfer to a substrate lysine. The process is triggered by PINK1, which phosphorylates ubiquitin on damaged mitochondria, which in turn recruits and activates Parkin. This leads to the ubiquitination of outer mitochondrial membrane proteins and clearance of the organelle. While the targets of Parkin on mitochondria are known, the factors determining substrate selectivity remain unclear. To investigate this, we examined how Parkin catalyzes ubiquitin transfer to substrates. We found that His433 in the RING2 domain catalyzes acyl transfer. In cells, mutation of His433 impairs mitophagy. In vitro ubiquitination assays with isolated mitochondria show that Mfn2 is a kinetically preferred substrate. Using proximity-ligation assays, we show that Mfn2 specifically co-localizes with PINK1 and phospho-ubiquitin in U2OS cells upon mitochondrial depolarization. We propose a model whereby ubiquitination of Mfn2 is efficient by virtue of its localization near PINK1, which leads to the recruitment and activation of Parkin via phospho-ubiquitin at these sites.