Arabidopsis thaliana and Saccharomyces cerevisiae NHX1 genes encode amiloride sensitive electroneutral Na+/H+ exchangers

Abstract

Sodium at high millimolar levels in the cytoplasm is toxic to plant and yeast cells. Sequestration of Na(+) ions into the vacuole is one mechanism to confer Na(+)-tolerance on these organisms. In the present study we provide direct evidence that the Arabidopsis thaliana At-NHX1 gene and the yeast NHX1 gene encode low-affinity electroneutral Na(+)/H(+) exchangers. We took advantage of the ability of heterologously expressed At-NHX1 to functionally complement the yeast nhx1-null mutant. Experiments on vacuolar vesicles isolated from yeast expressing At-NHX1 or NHX1 provided direct evidence for pH-gradient-energized Na(+) accumulation into the vacuole. A major difference between NHX1 and At-NHX1 is the presence of a cleavable N-terminal signal peptide (SP) in the former gene. Fusion of the SP to At-NHX1 resulted in an increase in the magnitude of Na(+)/H(+) exchange, indicating a role for the SP in protein targeting or regulation. Another distinguishing feature between the plant and yeast antiporters is their sensitivity to the diuretic compound amiloride. Whereas At-NHX1 was completely inhibited by amiloride, NHX1 activity was reduced by only 20-40%. These results show that yeast as a heterologous expression system provides a convenient model to analyse structural and regulatory features of plant Na(+)/H(+) antiporters.

The Arabidopsis Na+/H+Exchanger AtNHX1 Catalyzes Low Affinity Na+ and K+ Transport in Reconstituted Liposomes

“…The Arabidopsis AtNHX1 protein localizes to the vacuole, and Na ϩ /H ϩ exchange could be detected in tonoplast fractions from transgenic Arabidopsis overexpressing the protein (5). Na ϩ /H ϩ exchange activity could also be measured in vacuolar membrane fractions of yeast expressing the AtNHX1 protein (7). AtNHX1 shows greatest sequence homology with the ScNHX1 protein of Saccharomyces cerevisiae and the NHE transporters of mammalian cells (8,9).…”

Regulation of Vacuolar Na+/H+ Exchange in Arabidopsis thaliana by the Salt-Overly-Sensitive (SOS) Pathway

“…This is in contrast to amiloride, which did not affect Na ϩ /H ϩ exchange at any of the concentrations tested (data not shown). The inhibitory effects of amiloride and its analogs on AtNHX1 expressed in yeast and reconstituted in liposomes have been reported recently (18,19). In comparative studies of yeast cells expressing AtNHX1 from Arabidopsis and overexpressing NHX1 from yeast, Darley et al (18) showed that, activity originating from the Arabidopsis exchanger was completely inhibited with 120 M amiloride while activity originating from the yeast exchanger was inhibited 20 -40% at the same concentration.…”

“…When At-NHX1, an Arabidopsis homolog of the S. cerevisiae Na ϩ /H ϩ exchanger NHX1, was overexpressed in Arabidopsis, tonoplast exchange activity increased (relative to activity in wild-type plants) and the plants were better able to grow in high levels of salt (20,21). The transport activity of AtNHX1 has been demonstrated by expressing the gene in yeast (18), tomato (22), and Brassica (23) and by reconstituting it into liposomes (19). To understand how intracellular Na ϩ levels are regulated during salt stress, it will be necessary to determine how the individual proteins that transport Na ϩ function coordinately.…”

“…The transport activity of a plant tonoplast Na ϩ /H ϩ exchanger has been well characterized (3,4,18,19). In addition, the molecular mechanism underlying tonoplast Na ϩ compartmentation by the exchanger and its function in plant salt tolerance have been recently demonstrated (20,21).…”

AtCCX1 transports Na+ and K+ in Pitch pastoris

“…However, recently, CCXs in Arabidopsis were also found to be related to Na + compartmentation. Heterologous expression of AtCCX3 in yeast increased accumulation of Na + in yeast (Morris et al, 2008), similar to NHX1 to promote Na + uptake (Kinclova et al, 2003;Darley et al, 2000;Fukuda et al, 2011). When compared with NHX1, AtCCX3 simultaneously transported K + and Mn 2+ apart from Na + .…”