We conducted an analysis of the topology of AtNHX1, an Arabidopsis thaliana vacuolar Na(+)/H(+) antiporter. Several hydrophilic regions of the antiporter were tagged with a hemagglutinin epitope, and protease protection assays were conducted to determine the membrane topology of the antiporter by using yeast as a heterologous expression system. The overall structure of AtNHX1 is distinct from the human Na(+)/H(+) antiporter NHE1 or any known Na(+)/H(+) antiporter. It is comprised of nine transmembrane domains and a hydrophilic C-terminal domain. Three hydrophobic regions do not appear to span the tonoplast membrane, yet appear to be membrane associated. Our results also indicate that, whereas the N terminus of AtNHX1 is facing the cytosol, almost the entire C-terminal hydrophilic region resides in the vacuolar lumen. Deletion of the hydrophilic C terminus resulted in a dramatic increase in the relative rate of Na(+)/H(+) transport. The ratio of Na(+)/K(+) transport was twice that of the unmodified AtNHX1. This altered ratio resulted from a relatively small decrease in K(+)/H(+) transport with a large increase in Na(+)/H(+) transport. The vacuolar localization of the C terminus of the AtNHX1, taken together with the regulation of the antiporter selectivity by its C terminus, demonstrates the existence of luminal vacuolar regulatory mechanisms of the antiporter activity.
