Arabidopsis thaliana and Saccharomyces cerevisiae NHX1 genes encode amiloride sensitive electroneutral Na+/H+ exchangers.
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.
Darley
C P
CP
Department of Developmental Genetics, Vrije Universiteit, BioCentrum Amsterdam, De Boelelaan 1087, Amsterdam, 1081 HV, The Netherlands.
van Wuytswinkel
O C
OC
van der Woude
K
K
Mager
W H
WH
de Boer
A H
AH
D000584:Amiloride / Q000378:metabolism / Q000494:pharmacology*; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D027682:Cation Transport Proteins; D002934:Cinnamates; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004232:Diuretics / Q000378:metabolism / Q000494:pharmacology*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D005816:Genetic Complementation Test; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects; D010641:Phenotype; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000502:physiology; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012492:Salts / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism
D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D002934:Cinnamates; D004232:Diuretics; D005656:Fungal Proteins; C117751:NHX1 protein, Arabidopsis; C492183:NHX1 protein, S cerevisiae; D010940:Plant Proteins; D021382:Protein Sorting Signals; D029701:Saccharomyces cerevisiae Proteins; D012492:Salts; D017923:Sodium-Hydrogen Exchangers; D006921:Hygromycin B; C026273:hygromycin A; D000584:Amiloride; D006859:Hydrogen; D012964:Sodium