Identification of two essential arginine residues in UhpT, the sugar phosphate antipoter of Escherichia coli

M. C. Fann, A. H. Davies, A. Varadhachary, T. Kuroda, C. Sevier, T. Tsuchiya, P. C. Maloney

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39 Citations (Scopus)

Abstract

Three lines of evidence indicate that arginine-46 (R46) and arginine-275 (R275) are essential to the function of UhpT, the Pi-linked antiport protein of Escherichia coli. A role for arginine was initially suggested by the sensitivity of UhpT to inhibition by 2,3-butanedione, an arginine-directed probe. Since the presence of substrate protected against this inhibition, this work further suggested that arginine(s) may lie at or near the UhpT active site. In other work, each UhpT arginine was examined individually by using site-directed mutagenesis to generate a cysteine or a lysine derivative. With two exceptions (R46, R275), all arginines could be replaced by either cysteine (10 of 14 residues) or lysine (12 of 14) without loss of function, implicating R46 and R275 as essential to UhpT function. This idea was strengthened by examining a multiple alignment of the eleven known UhpT-related proteins (≤30% identity). That alignment showed R46 and R275 were two of the only three arginines strongly conserved in this group of proteins. Considered together, these different approaches lead us to conclude that UhpT and its relatives have only two arginine residues (R46, R275) whose presence is essential to function. Prior biochemical work had placed R275 at the external entrance to the translocation pathway, and a symmetry argument emerging from the multiple alignment suggests a similar position for R46. Accordingly, by virtue of their locations at the entrance to this pathway, we speculate that R46 and R275 function in establishing substrate specificity.

Original languageEnglish
Pages (from-to)187-195
Number of pages9
JournalJournal of Membrane Biology
Volume164
Issue number2
DOIs
Publication statusPublished - Jul 15 1998

Keywords

  • Butanedione
  • Membrane transporter protein
  • Phosphate-binding
  • Reconstitution

ASJC Scopus subject areas

  • Biophysics
  • Physiology
  • Cell Biology

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    Fann, M. C., Davies, A. H., Varadhachary, A., Kuroda, T., Sevier, C., Tsuchiya, T., & Maloney, P. C. (1998). Identification of two essential arginine residues in UhpT, the sugar phosphate antipoter of Escherichia coli. Journal of Membrane Biology, 164(2), 187-195. https://doi.org/10.1007/s002329900404