Genetic evidence for an essential oscillation of transmembrane-spanning segment 5 in the Escherichia coli ammonium channel AmtB.
Ammonium channels, called Amt or Mep, concentrate NH(4)(+) against a gradient. Each monomer of the trimer has a pore through which substrate passes and a C-terminal cytoplasmic extension. The importance of the C-terminal extension to AmtB activity remains unclear. We have described lesions in conserved C-terminal residues that inactivate AmtB and here characterize 38 intragenic suppressors upstream of the C terminus ( approximately 1/3 of total suppressors). Three that occurred repeatedly, including the previously characterized W148L at the pore entry, restored growth at low NH(3) to nearly wild-type levels and hence restored high activity. V116L completely restored function to two of the mutant proteins and, when separated from other lesions, did not damage wild-type AmtB. A179E notably altered folding of AmtB, compensated for all inactivating C-terminal lesions, and damaged wild-type AmtB. V116L and A179E lie at the cytoplasmic end of transmembrane-spanning segments (TM) 3 and 5, respectively, and the proximal part of the C-terminal tail makes intimate contacts with the loops following them before crossing to the adjacent monomer. Collectively, the properties of intragenic suppressor strains lead us to postulate that the C-terminal tail facilitates an oscillation of TM 5 that is required for coordinated pore function and high AmtB activity. Movement of TM 5 appears to control the opening of both the periplasmic entry and the cytoplasmic exit to the pore.
Inwood
William B
WB
Department of Plant and Microbial Biology, University of California, Berkeley, California 94720, USA.
Hall
Jason A
JA
Kim
Kwang-Seo
KS
Fong
Rebecca
R
Kustu
Sydney
S
D049069:ATP-Dependent Proteases / Q000378:metabolism; D000595:Amino Acid Sequence; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007553:Isotope Labeling; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000378:metabolism; D051922:PII Nitrogen Regulatory Proteins / Q000378:metabolism; D011487:Protein Conformation; D017384:Sequence Deletion; D013489:Suppression, Genetic
C111096:AmtB protein, E coli; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D051922:PII Nitrogen Regulatory Proteins; C497878:glnK protein, E coli; D009713:Nucleotidyltransferases; D049069:ATP-Dependent Proteases; C513383:FtsH protein, E coli