Topological analysis of a plant vacuolar Na ⁺ /H ⁺ antiporter reveals a luminal C terminus that regulates antiporter cation selectivity

Abstract

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.

Vacuolar Na + /H + antiporter cation selectivity is regulated by calmodulin from within the vacuole in a Ca 2+ - and pH-dependent manner

“…Analyses of the transcriptional profile of AtNHX1 insertional knockout mutant plants growing in the absence and presence of salt (14) revealed changes in gene expression supporting the notion that, as in the yeast ortholog Nhx1p (15), AtNHX1 plays a significant role in protein trafficking and protein targeting, probably via the regulation of intravesicular acidic pH (16). Topological analyses revealed that, whereas the N terminus of AtNHX1 is facing the cytosol, almost the entire C-terminal hydrophilic region of the protein resides in the vacuolar lumen (17). Moreover, the deletion of the C terminus of AtNHX1 doubled the Na ϩ ͞K ϩ selectivity ratio of the antiporter, suggesting a regulatory role of the C terminus of the antiporter (17).…”

“…N terminus and C terminus 3xFLAG-tagged AtCaM15 constructs were generated by a fusion PCR method (17) and cloned into the SpeI and PstI site of p425GPD and p425CYC1 plasmids (American Type Culture Collection). The resulting constructs were transformed into the yeast strain W303-1B (MAT␣ ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1 can1-100) independently.…”

“…The resulting constructs were transformed into the yeast strain W303-1B (MAT␣ ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1 can1-100) independently. Yeast vacuoles were isolated as reported earlier (17). Vacuoles (100 g of protein) were incubated with 10 g͞ml proteinase K in 10 mM Tris͞Mes, pH 6.9, in the presence or absence of 1% Triton X-100 for 30 min at room temperature.…”

“…AtNHX1 was tagged with a 3xHA tag at the C terminus in the p426TEF vector (pHpl-13) (17), and AtCaM15 and CaM81 (AtCaM7) were tagged with a 3xFLAG tag at the C terminus in p425GPD. The 3xHA tagged Cterminally truncated AtNHX1 (440 aa from N terminus) was generated by fusion PCR (17) and cloned into EcoRI͞SalI site of p426TEF. The resulting plasmids were introduced into the yeast strain TY002 (MAT␣ pep4-3 leu2 trp1 ura3-52 prb1-1122 nhx1⌬::TRP1 ena1-5⌬::KanMX6).…”

“…Topological analyses revealed that, whereas the N terminus of AtNHX1 is facing the cytosol, almost the entire C-terminal hydrophilic region of the protein resides in the vacuolar lumen (17). Moreover, the deletion of the C terminus of AtNHX1 doubled the Na ϩ ͞K ϩ selectivity ratio of the antiporter, suggesting a regulatory role of the C terminus of the antiporter (17). The vacuolar localization of the AtNHX1 C terminus, together with the regulation of the antiporter selectivity by its C terminus, implied that regulatory determinants of the antiporter activity could exert their effects from within the vacuole (17).…”