Development of a Novel Assay for Pink1-Parkin-Miro1 Pathway

Mutations in the parkin and PINK1 genes are a major cause of autosomal recessive early-onset Parkinson disease (PD). Several studies have shown that PINK1 phosphorylates Parkin, Parkin substrates, and ubiquitin, thereby stimulating ubiquitination of proteins on the mitochondrial surface (i.e. Miro1) by the Parkin E3 ligase. The ubiquitinated proteins then are degraded, which signals for mitochondrial clearance. Although it is imperative to understand these events at the molecular level, current biochemical methods are cumbersome and lack the resolution to answer these questions thoroughly. Most importantly, physiological E2 partners for Parkin ligase are contested in the field. Here, we report a novel assay to study the PINK1-Parkin-Miro1 pathway that fundamentally addresses the aforementioned problems. In our assay, the ubiquitin c-terminus is chemically activated as a thioester that can undergo transthiolation with the catalytic cysteine of the Parkin E3 ligase, thereby directly charging ubiquitin to Parkin E3s without the need for E1, E2 and ATP. We demonstrate that this simplified two-component system recapitulates two native functions of Parkin, Miro1 ubiquitination and autoubiquitination. This simplified E2-bypassing assay will be generally useful to study the Parkin mechanism and to screen small molecular Parkin activators to treat PD.