Mutants of the Arabidopsis thaliana cation/H+ antiporter AtNHX1 conferring increased salt tolerance in yeast: the endosome/prevacuolar compartment is a target for salt toxicity.
Mutants of the plant cation/H(+) antiporter AtNHX1 that confer greater halotolerance were generated by random mutagenesis and selected in yeast by phenotypic complementation. The amino acid substitutions that were selected were conservative and occurred in the second half of the membrane-associated N terminus. AtNHX1 complemented the lack of endogenous ScNHX1 in endosomal protein trafficking assays. Growth enhancement on hygromycin B and vanadate media agreed with a generally improved endosomal/prevacuolar function of the mutated proteins. In vivo measurements by (31)P NMR revealed that wild-type and mutant AtNHX1 transporters did not affect cytosolic or vacuolar pH. Surprisingly, when yeast cells were challenged with lithium, a tracer for sodium, the main effect of the mutations in AtNHX1 was a reduction in the amount of compartmentalized lithium. When purified and reconstituted into proteoliposomes or assayed in intact vacuoles isolated from yeast cells, a representative mutant transporter (V318I) showed a greater cation discrimination favoring potassium transport over that of sodium or lithium. Together, our data suggest that the endosome/prevacuolar compartment is a target for salt toxicity. Poisoning by toxic cations in the endosome/prevacuolar compartment is detrimental for cell functions, but it can be alleviated by improving the discrimination of transported alkali cations by the resident cation/H(+) antiporter.
Hernández
Agustín
A
Instituto de Recursos Naturales y Agrobiología, Consejo Superior de Investigaciones Científicas (IRNASE-CSIC), Avda. Reina Mercedes 10, Seville 41012, Spain.
Jiang
Xingyu
X
Cubero
Beatriz
B
Nieto
Pedro M
PM
Bressan
Ray A
RA
Hasegawa
Paul M
PM
Pardo
José M
JM
D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D002412:Cations; D002451:Cell Compartmentation / Q000187:drug effects; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism*; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D021381:Protein Transport / Q000187:drug effects; D011510:Proteolipids / Q000187:drug effects / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D055049:Salt Tolerance / Q000187:drug effects*; D012965:Sodium Chloride / Q000633:toxicity*; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000378:metabolism*; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism*
D029681:Arabidopsis Proteins; D027682:Cation Transport Proteins; D002412:Cations; C117751:NHX1 protein, Arabidopsis; D011510:Proteolipids; D017923:Sodium-Hydrogen Exchangers; C020485:proteoliposomes; D012965:Sodium Chloride