Synthetic tools to redirect the ubiquitin E3 ligase activity of, PRT1, a plant-specific N-recognin

The Arg/N-degron pathway regulates important agronomic traits. In plants, the PRT1 protein binds to aromatic amino-terminal residues and recruits ubiquitin (Ub) conjugating enzymes to ubiquitinate substrate proteins. Here we demonstrate that PRT1 recruits the UBC35-UEV1 complex via RING1 of PRT1. This stimulates UBC35 to produce K63-linked Ub chains. To afford chemical control over Ub signalling, we construct a synthetic substrate of PRT1 with an N-terminal calmodulin-binding peptide (CBP). The addition of the CBP sequence to the substrate creates an off switch for PRT1-dependent ubiquitination that is regulated by calcium and calmodulin. Finally, we produce a set of nanobodies that are recognised by PRT1 and induce the ubiquitination of a reporter protein. Altogether, we introduce and biochemically validate several new tools to both redirect and chemically control the Arg/N-degron pathway.