The C-terminal tail of the plant endosomal-type NHXs plays a key role in its function and stability.

Typically, Na/H antiporters (NHXs) possess a conserved N-terminus for cation binding and exchange and a hydrophilic C-terminus for regulating the antiporter activity. Plant endosomal-type NHXs play important roles in protein trafficking, as well as K and vesicle pH homeostasis, however the role of the C-terminal tail remains unclear. Here, the function of MnNHX6, an endosomal-type NHX in mulberry, was investigated using heterologous expression in yeast. Functional and localization analyses of C-terminal truncation and mutations in MnNHX6 revealed that the C-terminal conserved region was responsible for the function and stability of the protein and its hydrophobicity, which is a key domain requirement. Nuclear magnetic resonance spectroscopy provided direct structural evidence and yeast two-hybrid screening indicated that this functional domain was also necessary for interaction with sorting nexin 1. Our findings demonstrate that although the C-terminal tail of MnNHX6 is intrinsically disordered, the C-terminal conserved region may be an important part of the external mouth of this transporter, which controls protein function and stability by serving as an inter-molecular cork with a chain mechanism. These findings improve our understanding of the roles of the C-terminal tail of endosomal-type NHXs in plants and the ion transport mechanism of NHX-like antiporters.