The KtrA and KtrE subunits are required for Na+-dependent K+ uptake by KtrB across the plasma membrane in Synechocystis sp. strain PCC 6803.
The Na+-dependent K+ uptake KtrABE system is essential for the adaptation of Synechocystis to salinity stress and high osmolality. While KtrB forms the K+-translocating pore, the role of the subunits KtrA and KtrE for Ktr function remains elusive. Here, we characterized the role of KtrA and KtrE in Ktr-mediated K+ uptake and in modulating Na+ dependency. Expression of KtrB alone in a K+ uptake-deficient Escherichia coli strain conferred low K+ uptake activity that was not stimulated by Na+. Coexpression of both KtrA and KtrE with KtrB increased the K+ transport activity in a Na+-dependent manner. KtrA and KtrE were found to be localized to the plasma membrane in Synechocystis. Site-directed mutagenesis was used to analyze the role of single charged residues in KtrB for Ktr function. Replacing negatively charged residues facing the extracellular space with residues of the opposite charge increased the apparent Km for K+ in all cases. However, none of the mutations eliminated the Na+ dependency of Ktr-mediated K+ transport. Mutations of residues on the cytoplasmic side had larger effects on K+ uptake activity than those of residues on the extracellular side. Further analysis revealed that replacement of R262, which is well conserved among Ktr/Trk/HKT transporters in the third extracellular loop, by Glu abolished transport activity. The atomic-scale homology model indicated that R262 might interact with E247 and D261. Based on these data, interaction of KtrA and KtrE with KtrB increased the K+ uptake rate and conferred Na+ dependency.
Zulkifli
Lalu
L
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-07, Sendai 980-8579, Japan.
Akai
Masaro
M
Yoshikawa
Asuka
A
Shimojima
Mie
M
Ohta
Hiroyuki
H
Guy
H Robert
HR
Uozumi
Nobuyuki
N
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000235:genetics / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism*; D005816:Genetic Complementation Test; D008954:Models, Biological; D008969:Molecular Sequence Data; D011188:Potassium / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism*; D046939:Synechocystis / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D012964:Sodium; D011188:Potassium