Determination of the essentiality of the eight cysteine residues of the NrtA protein for high-affinity nitrate transport and the generation of a functional cysteine-less transporter

Unkles, S, Rouch, D, Wang, Y, Siddiqi, M, Okomoto, M, Stephenson, R, Kinghorn, J and Glass, A 2005, 'Determination of the essentiality of the eight cysteine residues of the NrtA protein for high-affinity nitrate transport and the generation of a functional cysteine-less transporter', Biochemistry, vol. 44, no. 14, pp. 5471-5477.


Document type: Journal Article
Collection: Journal Articles

Title Determination of the essentiality of the eight cysteine residues of the NrtA protein for high-affinity nitrate transport and the generation of a functional cysteine-less transporter
Author(s) Unkles, S
Rouch, D
Wang, Y
Siddiqi, M
Okomoto, M
Stephenson, R
Kinghorn, J
Glass, A
Year 2005
Journal name Biochemistry
Volume number 44
Issue number 14
Start page 5471
End page 5477
Total pages 7
Publisher American Chemical Society
Abstract All eight cysteine residues, C90, C94, C143, C147, C219, C325, C367, and C431, present in transmembrane domains of the Aspergillus nidulans NrtA nitrate transporter protein were altered individually by site-specific mutagenesis. The results indicate that six residues, C90, C147, C219, C325, C367, and C431, are not required for nitrate transport. Although alterations of C94 and C143 are less well tolerated, these residues are not mandatory and their possible role is discussed. A series of constructs, all completely devoid of cysteine residues, was generated to permit future cysteine-scanning mutagenesis. The optimum cysteine-less combination was identified as C90A, C94A, C143A, C147T, C219S, C325S, C367S, and C431S. This mutant combination yielded transformant strains with up to 40% of wild-type nitrate transport activity. Furthermore, the K m value and the level of protein expression were found to be similar to those of the wild-type. This cysteine-less vector should allow us to investigate in detail potentially interesting NrtA amino acids (e.g. identified from homology comparisons) which may be involved in transport, by altering these singly to cysteine and studying such residues by thiol chemistry.
Subject Receptors and Membrane Biology
DOI - identifier 10.1021/bi047732e
ISSN 0006-2960
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