Distinct phosphorylation signals drive acceptor versus self-ubiquitination selection by Parkin
The RBR E3 ligase parkin is recruited to the outer mitochondrial membrane (OMM) during oxidative stress where it becomes activated and ubiquitinates numerous proteins. Parkin activation involves binding of a phosphorylated ubiquitin (pUb), followed by phosphorylation of parkin itself, both mediated by the OMM kinase, PINK1. However, targeted mitochondrial proteins have little structural or sequence similarity, with the commonality between substrates being proximity to the OMM. Here, we demonstrate that parkin efficiently ubiquitinates a mitochondrial acceptor pre-ligated to pUb and phosphorylation of parkin triggers autoubiquitination activity. Mitochondrial target proteins, Miro1 or CISD1, tethered to pUb are ubiquitinated by parkin more efficiently than if alone or Ub-tethered and ubiquitin molecules are ligated to acceptor protein lysines and not pUb. Parkin phosphorylation is not required for acceptor-pUb ubiquitination. In fact, only phospho-parkin induced self-ubiquitination and deletion of Ubl or mutation at K211N inhibited self-ubiquitination. We propose divergent parkin mechanisms whereby parkin-mediated ubiquitination of acceptor proteins is driven by binding to pre-existing pUb and subsequent parkin phosphorylation triggers autoubiquitination. This finding is critical for understanding parkins role in mitochondrial homeostasis and has implications on targets for therapeutics.