Phosphorylation by CIPK23 regulates the high-affinity Mn transporter NRAMP1 in Arabidopsis

Manganese (Mn) is essential for plants but is toxic when taken up in excess. To maintain Mn homeostasis, the root Mn transporter natural resistance associated macrophage protein 1 (NRAMP1) cycles from the plasma membrane to endosomes upon phosphorylation. To identify the kinase involved, a split-luciferase screening was carried out between NRAMP1 and kinases of the CIPK family and identified CIPK23 as a partner of NRAMP1. The interaction was confirmed by split-mCitrine bimolecular fluorescence complementation and co-immunoprecipitation assays. In vitro phosphorylation assays pinpointed two CIPK23 target residues in NRAMP1, among which serine 20, important for endocytosis. Interestingly, Mn-induced internalization of NRAMP1 was unaffected by cipk23 mutation suggesting a potential redundancy between CIPK23 and other kinase(s). How CIPK23 could regulate NRAMP1 in response to Mn availability is discussed.