Identification of amino acid substitutions that toggle substrate selectivity of the yeast arsenite transporter Acr3.

ABSTRACT

The Acr3 protein family plays a crucial role in metalloid detoxification and includes members from bacteria to higher plants. Most of the Acr3 transporters studied so far are specific for arsenite, whereas Acr3 from budding yeast also shows some capacity to transport antimonite. However, the molecular basis of Acr3 substrate specificity remains poorly understood. By analyzing randomly generated and rationally designed yeast Acr3 variants, critical residues determining substrate specificity were identified for the first time. Replacement of Val173 with Ala abolished antimonite transport without affecting arsenite extrusion. In contrast, substitution of Glu353 with Asp resulted in a loss of arsenite transport activity and a concomitant increase in antimonite translocation capacity. Importantly, Val173 is located close to the hypothetical substrate binding site, whereas Glu353 has been proposed to participate in substrate binding. Identification of key residues conferring substrate selectivity provides a valuable starting point for further studies of the Acr3 family and may have implications for the development of biotechnological applications in metalloid remediation. Moreover, our data contribute to understanding why members of the Acr3 family evolved as arsenite-specific transporters in an environment of ubiquitously present arsenic and trace amounts of antimony.