Oligomerization of NhaA, the Na+/H+ antiporter of Escherichia coli in the membrane and its functional and structural consequences.
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.
Division of Microbial and Molecular Ecology, Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Y
Y
Gerchman
A
A
Rimon
M
M
Venturi
E
E
Padan
D000483:Alleles; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D021961:Cell Membrane Structures / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D003432:Cross-Linking Reagents / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005804:Genes, Lethal; D005816:Genetic Complementation Test; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D013329:Structure-Activity Relationship; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C102839:NhaA protein, E coli; D010455:Peptides; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers