PMID:11258962
| Citation |
Gerchman, Y, Rimon, A, Venturi, M and Padan, E (2001) Oligomerization of NhaA, the Na+/H+ antiporter of Escherichia coli in the membrane and its functional and structural consequences. Biochemistry 40:3403-12 |
|---|---|
| Abstract |
Recently, a two-dimensional crystal structure of NhaA, the Na+/H+ antiporter of Escherichia coli has been obtained [Williams, K. A., Kaufer, U. G., Padan, E., Schuldiner, S. and Kühlbrandt, W. (1999) EMBO J., 18, 3558-3563]. In these crystals NhaA exists as a dimer. Using biochemical and genetic approaches here we show that NhaA exists in the native membrane as a homooligomer. Functional complementation between the polypeptides of NhaA was demonstrated by coexpression of pairs of conditional lethal (at high pH in the presence of Na+) mutant alleles of nhaA in EP432, a strain lacking antiporters. Physical interaction in the membrane was shown between the His-tagged NhaA polypeptide which is readily affinity purified from DM-solubilized membranes with a Ni2+-NTA column and another which is not; only when coexpressed did both copurify on the column. The organization of the oligomer in the membrane was studied in situ by site-directed cross-linking experiments. Cysteine residues were introduced--one per NhaA--into certain loops of Cys-less NhaA, so that only intermolecular cross-linking could take place. Different linker-size cross-linkers were applied to the membranes, and the amount of the cross-linked protein was analyzed by mobility shift on SDS-PAGE. The results are consistent with homooligomeric NhaA and the location of residue 254 in the interface between monomers. Intermolecular cross-linking of V254C caused an acidic shift in the pH profile of NhaA. |
| Links | |
| Keywords |
Alleles; Bacterial Proteins/biosynthesis; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Bacterial Proteins/ultrastructure; Cell Membrane/chemistry; Cell Membrane/genetics; Cell Membrane/metabolism; Cell Membrane/ultrastructure; Cell Membrane Structures/chemistry; Cell Membrane Structures/genetics; Cell Membrane Structures/metabolism; Cell Membrane Structures/ultrastructure; Cross-Linking Reagents/chemistry; Escherichia coli/genetics; Escherichia coli/growth & development; Escherichia coli/metabolism; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Genes, Lethal; Genetic Complementation Test; Mutagenesis, Site-Directed; Peptides/chemistry; Peptides/genetics; Peptides/metabolism; Plasmids/chemistry; Plasmids/metabolism; Recombinant Proteins/biosynthesis; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Recombinant Proteins/ultrastructure; Sodium-Hydrogen Antiporter/biosynthesis; Sodium-Hydrogen Antiporter/genetics; Sodium-Hydrogen Antiporter/metabolism; Sodium-Hydrogen Antiporter/ultrastructure; Structure-Activity Relationship; Transformation, Genetic |
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