Abstract
The CorA transport system is the major Mg2+ influx pathway for bacteria and the Archaea. CorA contains three C-terminal transmembrane segments. No conserved charged residues are apparent within the membrane, suggesting that Mg2+ influx does not involve electrostatic interactions. We have mutated conserved residues within the third transmembrane segment to identify sites involved in transport. Mutation of conserved aromatic residues at either end of the membrane segment to alternative aromatic amino acids did not affect total cation uptake or cation affinity. Mutation to alanine greatly diminished uptake with little change in cation affinity implying that the conserved aromatic residues play a structural role in stabilizing this membrane segment of CorA at the interface between the bilayer and the aqueous environment. In contrast, mutation of Tyr292, Met299, and Tyr307 greatly altered the transport properties of CorA. Y292F, Y292S, Y292C, or Y292I mutations essentially abolished transport, without effect on expression or membrane insertion. M299C and M299A mutants exhibited a decrease in cation affinity for Mg2+, Co2+, or Ni2+ of 10-50-fold without a significant change in uptake capacity. Mutations at Tyr307 had no significant effect on cation uptake capacity; however, the affinity of Y307F and Y307A mutations for Mg2+ and Co2+ was decreased 3-10-fold, while affinity for Ni2+ was unchanged compared with the wild type CorA. In contrast, the affinity of the Y307S mutant for all three cations was decreased 2-5-fold. Projection of the third transmembrane segment as an alpha-helix suggests that Tyr292, Met299, and Tyr307 all reside on the same face of the alpha-helix. We interpret the transport data to suggest that a hydroxyl group is important at Tyr307, and that these three residues interact with Mg2+ during transport, forming part of the cation pore or channel within CorA.
Highlights
The CorA transport system is the major Mg2؉ influx pathway for bacteria and the Archaea
In the S. typhimurium CorA, Glu251 is the only negative charge within the membrane, but a negative charge at this or nearby positions is not conserved in the other CorA homologs [5]
The most charge dense of the biological cations, Mg2ϩ might be expected to interact with negatively charged amino acids in passing through the membrane
Summary
All media were obtained from Difco (Detroit, MI). All other reagents were from Sigma unless otherwise specified. Uptake was terminated after 5 min by addition of 5 ml of ice-cold transport wash buffer (N-minimal medium without glucose and casamino acids, with 5 mM Mg2ϩ and 1 mM EDTA). The wild type affinity of CorA for Mg2ϩ, Ni2ϩ, and Co2ϩ was about 20, 300, and 30 M, respectively In these assays, unless otherwise indicated, the Ni2ϩ concentration was set at 200 M. Unless otherwise indicated, the Ni2ϩ concentration was set at 200 M This is approximately equal to the Ka for Ni2ϩ uptake by the wild type CorA transporter and is roughly comparable to the Km for an enzyme. If the Ka for Ni2ϩ is changed by a mutation, the Vmax or maximal uptake capacity cannot be directly compared with that of the wild type transporter. Apparent cation affinities between experiments did not vary more than 3-fold for the same mutant allele
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