N-degron pathway of proteolysis conducts MtERF75 protein stability
ERF-VII proteins have a characteristic N-terminus (N-degron) with a cysteine (Cys) residue at the second position that leads to specific degradation by the N-degron-dependent ubiquitin proteasome system (UPS) in Arabidopsis. We tested whether the N-terminal part of MtERF conduct to targeted degradation by the N-degron pathway of proteolysis. Two constructions comprising the first 80 amino acids of MtERF75 were fused to the luciferase sequence (LUC). One version (MC80_ERF75) was characterized by the N-terminal Cys residue in MtERF75, whereas in MA80_ERF75 the Cys residue was replaced by an Ala that is insensitive to O2. The measurement of the activity of LUC reveals the presence and thus the stability of the protein. The MC /MA80_ERF75: LUC constructs were tested in A. thaliana protoplasts of wild type (Col-0) (Fig. 7a) and a mutant line lacking the E3 ligase (prt6 ) involved in the N-degron pathway of proteolysis (Fig. 7b). Experiments with the MA:80_ERF75: LUC construct in Col-0 protoplasts showed a significant increase in luciferase activity compared with MC80_ERF75: LUC, indicating the destabilizing role of Cys. Similarly, the luciferase signal was also higher in protoplasts of prt6plants compared with Col-0, indicating that the stability of the MC80_MtERF75: LUC chimeric protein is regulated by the N-degron pathway.