Distinct phosphorylation signals drive acceptor versus self-ubiquitination selection by Parkin

Abstract

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 bind ing of a phosphorylated ubiquitin (pUb), followed by phosphorylation of parkin itself, both mediated by the OMM kinase, PINK1. However, targeted mitochon drial proteins have little structural or sequence sim ilarity, with the commonality between substrates be ing proximity to the OMM. Here, we demonstrate that parkin efficiently ubiquitinates a mitochondrial accep tor 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 phos phorylation is not required for acceptor-pUb ubiqui tination. In fact, only phospho-parkin induced self ubiquitination and deletion of Ubl or mutation at K211N inhibited self-ubiquitination.