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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amiloride / metabolism
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Amiloride / pharmacology*
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Arabidopsis / genetics*
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Arabidopsis / metabolism
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Arabidopsis Proteins*
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Carrier Proteins / chemistry
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Carrier Proteins / genetics
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Carrier Proteins / metabolism*
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Cation Transport Proteins*
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Cinnamates*
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Cloning, Molecular
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Conserved Sequence
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Diuretics / metabolism
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Diuretics / pharmacology*
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Fungal Proteins / chemistry
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Fungal Proteins / genetics
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Fungal Proteins / metabolism
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Gene Deletion
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Genetic Complementation Test
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Hydrogen / metabolism
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Hydrogen-Ion Concentration
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Hygromycin B / analogs & derivatives
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Hygromycin B / pharmacology
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Inhibitory Concentration 50
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Intracellular Membranes / drug effects
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Intracellular Membranes / metabolism
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Ion Transport / drug effects
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Phenotype
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Plant Proteins / chemistry
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Plant Proteins / genetics
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Plant Proteins / metabolism
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Protein Binding
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Protein Sorting Signals / physiology
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Protein Structure, Tertiary
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Saccharomyces cerevisiae / cytology
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Saccharomyces cerevisiae / genetics*
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins*
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Salts / pharmacology
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Sodium / metabolism
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Sodium / pharmacology
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Sodium-Hydrogen Exchangers / chemistry
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Sodium-Hydrogen Exchangers / genetics
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Sodium-Hydrogen Exchangers / metabolism*
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Vacuoles / drug effects
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Vacuoles / metabolism
Substances
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Arabidopsis Proteins
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Carrier Proteins
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Cation Transport Proteins
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Cinnamates
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Diuretics
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Fungal Proteins
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NHX1 protein, Arabidopsis
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NHX1 protein, S cerevisiae
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Plant Proteins
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Protein Sorting Signals
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Saccharomyces cerevisiae Proteins
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Salts
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Sodium-Hydrogen Exchangers
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Hygromycin B
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hygromycin A
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Amiloride
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Hydrogen
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Sodium