Partial deletion of a loop region in the high affinity K+ transporter HKT1 changes ionic permeability leading to increased salt tolerance.
HKT1 is a high affinity K(+) transporter protein that is a member of a large superfamily of transporters found in plants, bacteria, and fungi. These transporters are primarily involved in K(+) uptake and are energized by Na(+) or H(+). HKT1 is energized by Na(+) but also mediates low affinity Na(+) uptake and may therefore be a pathway for Na(+) uptake, which is toxic to plants. The aim of this study was to identify regions of HKT1 that are involved in K(+)/Na(+) selectivity and alter the amino acid composition in those regions to increase the ionic selectivity of the transporter. A highly charged loop was identified, and two deletions were created that resulted in the removal of charged and uncharged amino acids. The functional changes caused by the deletions were studied in yeast and Xenopus oocytes. The deletions improved the K(+)/Na(+) selectivity of the transporter and increased the salt tolerance of the yeast cells in which they were expressed. In light of recent structural models of members of this symporter superfamily, it was necessary to determine the orientation of this highly charged loop. Introduction of an epitope tag allowed us to demonstrate that this loop faces the outside of the membrane where it is likely to facilitate the interaction with cations such as K(+) and Na(+). This study has identified an important structural feature in HKT1 that in part determines its K(+)/Na(+) selectivity. Understanding the structural basis of the functional characteristics in transporters such as HKT1 may have important implications for increasing the salt tolerance of higher plants.
Liu
W
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CSIRO Plant Industry, Horticulture Unit, P.O. Box 350, Glen Osmond, SA 5064, Australia.
Schachtman
D P
DP
Zhang
W
W
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000502:physiology; D002463:Cell Membrane Permeability / Q000502:physiology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000502:physiology; D005260:Female; D066298:In Vitro Techniques; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D010940:Plant Proteins / Q000737:chemistry / Q000502:physiology*; D011188:Potassium / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000502:physiology*; D017384:Sequence Deletion; D012964:Sodium / Q000378:metabolism; D027981:Symporters; D014982:Xenopus laevis
D002352:Carrier Proteins; D027682:Cation Transport Proteins; C088864:HKT1 protein, plant; D008565:Membrane Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D027981:Symporters; D012964:Sodium; D011188:Potassium