PI-(3,5)P2-mediated oligomerization of the endosomal sodium/proton exchanger NHE9

Na ⁺ /H ⁺ exchangers are found in all cells to regulate intracellular pH, sodium levels and cell volume. Na ⁺ /H ⁺ exchangers are physiological homodimers that operate by an elevator alternating-access mechanism. While the structure of the core ion translocation domain is fairly conserved, the scaffold domain and oligomerization show larger structural variation. The Na ⁺ /H ⁺ exchanger NhaA from E. coli has a weak oligomerization interface mediated by a β-hairpin domain and homodimerization was shown to be dependent of the lipid cardiolipin. Organellar Na ⁺ /H ⁺ exchangers NHE6, NHE7 and NHE9 are likewise predicted to contain β-hairpin domains and a recent analysis of horse NHE9 indicated that the lipid PIP ₂ binds at the dimerization interface. Despite predicted lipid-mediated oligomerization, their structural validation has been lacking. Here, we report cryo-EM structures of E. coli NhaA and horse NHE9 with the coordination of cardiolipin and PI(3,5)P ₂ binding at the dimer interface, respectively. Cell based assays confirms that NHE9 is inactive at the plasma membrane and thermal-shift assays, solid-supported membrane (SSM) electrophysiology and MD simulations, corroborates that NHE9 specifically binds the endosomal PI(3,5)P ₂ lipid, which stabilizes the homodimer and enhances activity. Taken together, we propose specific lipids regulate Na ⁺ /H ⁺ exchange activity by stabilizing oligomerization and stimulating Na ⁺ binding under lipid-specific cues.