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  Oligomerization of NhaA, the Na+/H+ Antiporter of Escherichia coli in the Membrane and Its Functional and Structural Consequences

Gerchman, Y., Rimon, A., Venturi, M., & Padan, E. (2001). Oligomerization of NhaA, the Na+/H+ Antiporter of Escherichia coli in the Membrane and Its Functional and Structural Consequences. Biochemistry, 40(11), 3403-3412. doi:10.1021/bi002669o.

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 Creators:
Gerchman, Yoram1, Author
Rimon, Abraham1, Author
Venturi, Miro2, Author           
Padan, Etana1, Author
Affiliations:
1Division of Microbial and Molecular Ecology, Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel, ou_persistent22              
2Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068290              

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Free keywords: Peptides and proteins; onomers; ligomers; embranes; ucleic acid structure
 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 introducedone per NhaAinto 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.

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Language(s): eng - English
 Dates: 2001-01-032000-11-202001-02-172001-03-01
 Publication Status: Published in print
 Pages: 10
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1021/bi002669o
 Degree: -

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Title: Biochemistry
Source Genre: Journal
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Publ. Info: Columbus, Ohio : American Chemical Society
Pages: - Volume / Issue: 40 (11) Sequence Number: - Start / End Page: 3403 - 3412 Identifier: ISSN: 0006-2960
CoNE: https://pure.mpg.de/cone/journals/resource/954925384103