Cation Chloride Cotransporter 1 (CCC1) regulates pH and ionic conditions in the TGN/EE and is required for endomembrane trafficking

The secretory and endocytic pathways intersect at the Trans-Golgi Network/Early Endosome (TGN/EE). TGN/EE function depends on luminal pH adjustment, which is regulated by the combined activity of a proton pump and several proton/ion antiporters. The identity of the proton pump is known, as well the antiporters that catalyse cation and anion import into the TGN/EE. However, the protein(s) required to complete the transport circuit, and that mediates cation and anion efflux has not been identified. Here, we characterise Arabidopsis Cation Chloride Cotransporter (AtCCC1) and show that it is localised in the TGN/EE. We further demonstrate that regulation of both luminal pH and ion concentrations are dependent on AtCCC1 function, using pharmacological treatments and genetically encoded fluorescent sensors. Loss of AtCCC1 leads to alterations in cellular functions dependent on the TGN/EE including endo- and exocytosis, trafficking to the vacuole and trafficking of the plasma membrane protein PIN2. This discovery provides the cellular role for CCC1s and can explain the multitude of phenotypic defects observed in loss-of-function plants. Collectively, our results demonstrate that non-proton-coupled ion transport contributes to the regulation of TGN/EE luminal ionic and pH conditions; and that CCC1 is an essential missing component of the TGN/EE ion transport circuit.