Abstract
Metal ion transport by DCT1, a member of the natural resistance-associated macrophage protein family, is driven by protons. The stoichiometry of the proton to metal ion is variable, and under optimal transport conditions, more than 10 protons are co-transported with a single metal ion. To understand this phenomenon better, we used site-directed mutagenesis of DCT1 and analyzed the mutants by complementation of yeast suppressor of mitochondria import function-null mutants and electrophysiology with Xenopus oocytes. The mutation F227I resulted in an increase of up to 14-fold in the ratio between metal ions to protons transported. This observation suggests that low metal ion to proton transport of DCT1 resulting from a proton slippage is not a necessity of the transport mechanism in which positively charged protons are driving two positive charges of the metal ion in the same direction. It supports the idea that the proton slippage has a physiological advantage, and the proton slip was positively selected during the evolution of DCT1.
Highlights
Metal ions are vital elements for all living cells
It has been shown that DCT1 and Smf1p function as general divalent metal ion transporters in mammals and yeast, respectively
The most striking one is in the slips generated by the transporters, Hϩ slip through DCT1 and Naϩ slip through Smf1p [11]
Summary
Metal ions are vital elements for all living cells. The NRAMP1 family of metal ion transporters apparently plays a major role in metal ion homeostasis [1,2,3,4]. Xenopus oocytes have a very low metal ion uptake background, which makes them the ideal heterologous expression system for metal ion transporters. On changing the membrane potential from ϩ10 to Ϫ80 mV at this low pH, the number of Hϩ ions transported with one Fe2ϩ ion increased from 3 to ϳ18 [9]. In contrast to DCT1, Smf1p showed a metal ion-independent sodium slip through the proton-translocating pathway [9, 12]. The mechanism of this phenomenon is not well characterized, and the sites on the transporters that generate it are not known. We addressed the coupling between proton and metal ion transport, and report on a mutation in DCT1 that exhibits an ϳ14-fold increase in the ratio of metal ion to proton transport
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