HKT1 mediates sodium uniport in roots. Pitfalls in the expression of HKT1 in yeast.
The function of HKT1 in roots is controversial. We tackled this controversy by studying Na+ uptake in barley (Hordeum vulgare) roots, cloning the HvHKT1 gene, and expressing the HvHKT1 cDNA in yeast (Saccharomyces cerevisiae) cells. High-affinity Na+ uptake was not detected in plants growing at high K+ but appeared soon after exposing the plants to a K(+)-free medium. It was a uniport, insensitive to external K+ at the beginning of K+ starvation and inhibitable by K+ several hours later. The expression of HvHKT1 in yeast was Na+ (or K+) uniport, Na(+)-K+ symport, or a mix of both, depending on the construct from which the transporter was expressed. The Na+ uniport function was insensitive to external K+ and mimicked the Na+ uptake carried out by the roots at the beginning of K+ starvation. The K+ uniport function only took place in yeast cells that were completely K+ starved and disappeared when internal K+ increased, which makes it unlikely that HvHKT1 mediates K+ uptake in roots. Mutation of the first in-frame AUG codon of HvHKT1 to CUC changed the uniport function into symport. The expression of the symport from either mutants or constructs keeping the first in-frame AUG took place only in K(+)-starved cells, while the uniport was expressed in all conditions. We discuss here that the symport occurs only in heterologous expression. It is most likely related to the K+ inhibitable Na+ uptake process of roots that heterologous systems fail to reproduce.
Haro
Rosario
R
Departamento de Biotecnología, Universidad Politécnica de Madrid, 28040 Madrid, Spain.
Bañuelos
María A
MA
Senn
María E
ME
Barrero-Gil
Javier
J
Rodríguez-Navarro
Alonso
A
D000595:Amino Acid Sequence; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D017343:Genes, Plant / Q000235:genetics; D001467:Hordeum / Q000254:growth & development / Q000378:metabolism; D017136:Ion Transport; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018517:Plant Roots / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D036226:Seedlings / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D027981:Symporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014908:Triticum / Q000378:metabolism
D027682:Cation Transport Proteins; D018076:DNA, Complementary; C088864:HKT1 protein, plant; D010940:Plant Proteins; D027981:Symporters; D012964:Sodium; D011188:Potassium