PIP₂-mediated oligomerization of the endosomal sodium/proton exchanger NHE9.

Abstract

The strict exchange of Na⁺ for H⁺ ions across cell membranes is a reaction carried out in almost every cell. Na⁺/H⁺ exchangers that perform this task are physiological homodimers, and whilst the ion transporting domain is highly conserved, their dimerization differs. The Na⁺/H⁺ exchanger NhaA from Escherichia coli has a weak dimerization interface mediated by a β-hairpin domain and with dimer retention dependent on cardiolipin. Similarly, organellar Na⁺/H⁺ exchangers NHE6, NHE7 and NHE9 also contain β-hairpin domains and recent analysis of Equus caballus NHE9 indicated PIP₂ lipids could bind at the dimer interface. However, structural validation of the predicted lipid-mediated oligomerization has been lacking. Here, we report cryo-EM structures of E. coli NhaA and E. caballus NHE9 in complex with cardiolipin and phosphatidylinositol-3,5-bisphosphate PI(3,5)P₂ lipids binding at their respective dimer interfaces. We further show how the endosomal specific PI(3,5)P₂ lipid stabilizes the NHE9 homodimer and enhances transport activity. Indeed, we show that NHE9 is active in endosomes, but not at the plasma membrane where the PI(3,5)P₂ lipid is absent. Thus, specific lipids can regulate Na⁺/H⁺ exchange activity by stabilizing dimerization in response to either cell specific cues or upon trafficking to their correct membrane location.