Mutational analysis of the intramembranous H10 loop of yeast Nhxl reveals a critical role in ion homoeostasis and vesicle trafficking

Yeast Nhx1 [Na+(K+)/H+ exchanger 1] is an intracellular Na+(K+)/H+ exchanger, localizing to the late endosome where it is important for ion homoeostasis and vesicle trafficking. Phylo-genetic analysis of NHE (Na+/H+ exchanger) sequences has ident-ified orthologous proteins, including HsNHE6 (human NHE6), HsNHE7 and HsNHE9 of unknown physiological role. These ap-pear distinct from well-studied mammalian plasma membrane iso-forms (NHE1–NHE5). To explore the differences between plasma membrane and intracellular NHEs and understand the link be-tween ion homoeostasis and vesicle trafficking, we examined the consequence of replacing residues in the intramembranous H10 loop of Nhx1 between transmembrane segments 9 and 10. The critical role for the carboxy group of Glu355 in ion transport is con-sistent with the invariance of this residue in all NHEs. Surpris-ingly, residues specifically conserved in the intracellular isoforms (such as Phe357 and Tyr361) could not be replaced with closely similar residues (leucine and phenylalanine) found in the plasma membrane isoforms without loss of function, revealing un-expected side chain specificity. The trafficking phenotypes of all Nhx1 mutants, including hygromycin-sensitivity and missorting of carboxypeptidase Y, were found to directly correlate with pH homoeostasis defects and could be proportionately corrected by titration with weak base. The present study demonstrates the im-portance of the H10 loop of the NHE family, highlights the dif-ferences between plasma membrane and intracellular isoforms and shows that trafficking defects are tightly coupled with pH homoeostasis. Key words: endosomal pH, ion homoeostasis, Na+/H+ exchanger (NHE), salt tolerance, vesicle trafficking, yeast Na+(K+)/H+ exchanger 1 (Nhx1)