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

Mutations in Parkin and PINK1 cause early-onset familial Parkinson's disease. Parkin is a RING-In-Between-RING E3 ligase that transfers ubiquitin from an E2 enzyme to a substrate in two steps: (i) thioester intermediate formation on Parkin and (ii) 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 catalyses ubiquitin transfer to substrates. We found that His433 in the RING2 domain contributes to the catalysis of acyl transfer. In cells, the 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 (pUb) 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 pUb at these sites.