Topological analysis of a plant vacuolar Na+/H+ antiporter reveals a luminal C terminus that regulates antiporter cation selectivity.
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.
Yamaguchi
Toshio
T
Department of Pomology, University of California, Davis, CA 95616.
Apse
Maris P
MP
Shi
Huazhong
H
Blumwald
Eduardo
E
D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D007700:Kinetics; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism
D029681:Arabidopsis Proteins; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers