PMID:19884311
| Citation |
Inwood, WB, Hall, JA, Kim, KS, Fong, R and Kustu, S (2009) Genetic evidence for an essential oscillation of transmembrane-spanning segment 5 in the Escherichia coli ammonium channel AmtB. Genetics 183:1341-55 |
|---|---|
| Abstract |
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. |
| Links |
PubMed PMC2787425 Online version:10.1534/genetics.109.109579 |
| Keywords |
ATP-Dependent Proteases/metabolism; Amino Acid Sequence; Cation Transport Proteins/chemistry; Cation Transport Proteins/genetics; Cation Transport Proteins/metabolism; Cell Membrane/metabolism; Electrophoresis, Polyacrylamide Gel; Escherichia coli/cytology; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Isotope Labeling; Models, Molecular; Molecular Sequence Data; Nucleotidyltransferases/metabolism; PII Nitrogen Regulatory Proteins/metabolism; Protein Conformation; Sequence Deletion; Suppression, Genetic |
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