Gain of function mutations in membrane region M2C2 of KtrB open a gate controlling K+ transport by the KtrAB system from Vibrio alginolyticus.
KtrB, the K(+)-translocating subunit of the Na(+)-dependent bacterial K(+) uptake system KtrAB, consists of four M(1)PM(2) domains, in which M(1) and M(2) are transmembrane helices and P indicates a p-loop that folds back from the external medium into the cell membrane. The transmembrane stretch M(2C) is, with its 40 residues, unusually long. It consists of three parts, the hydrophobic helices M(2C1) and M(2C3), which are connected by a nonhelical M(2C2) region, containing conserved glycine, alanine, serine, threonine, and lysine residues. Several point mutations in M(2C2) led to a huge gain of function of K(+) uptake by KtrB from the bacterium Vibrio alginolyticus. This effect was exclusively due to an increase in V(max) for K(+) transport. Na(+) translocation by KtrB was not affected. Partial to complete deletions of M(2C2) also led to enhanced V(max) values for K(+) uptake via KtrB. However, several deletion variants also exhibited higher K(m) values for K(+) uptake and at least one deletion variant, KtrB(Delta326-328), also transported Na(+) faster. The presence of KtrA did not suppress any of these effects. For the deletion variants, this was due to a diminished binding of KtrA to KtrB. PhoA studies indicated that M(2C2) forms a flexible structure within the membrane allowing M(2C3) to be directed either to the cytoplasm or (artificially) to the periplasm. These data are interpreted to mean (i) that region M(2C2) forms a flexible gate controlling K(+) translocation at the cytoplasmic side of KtrB, and (ii) that M(2C2) is required for the interaction between KtrA and KtrB.
Hänelt
Inga
I
Abteilung Mikrobiologie, University of Osnabrück, D-49076 Osnabrück, Germany. haenelt@biologie.uni-osnabrueck.de
Löchte
Sara
S
Sundermann
Lea
L
Elbers
Katharina
K
Vor der Brüggen
Marc
M
Bakker
Evert P
EP
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001692:Biological Transport; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D011188:Potassium / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D044149:Vibrio alginolyticus / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D027682:Cation Transport Proteins; C113206:KtrB protein, Bacteria; D012964:Sodium; D011188:Potassium