Hyperactive mutation occurs adjacent to the essential glutamate 286 for transport in the yeast tryptophan permease Tat2.

In Saccharomyces cerevisiae, high-affinity tryptophan import is mediated by the plasma membrane permease Tat2. Herein, we identified hyperactive Tat2 mutations, I285V and I285T, which allowed the cells to grow at very low tryptophan concentrations (<4 μg/mL). The K_m value of wild-type Tat2 for tryptophan appeared to be 24 μg/mL, whereas that of Tat2^I285V and Tat2^I285T was 17 and 11 μg/mL, respectively. Normalized values of V_max/K_m for Tat2^I285V- and Tat2^I285T-mediated tryptophan import were 2-fold higher than that for Tat2, suggesting that these mutations increase the affinity for tryptophan, and mediate transport at very low tryptophan concentrations. I285 resides adjacent to E286, a fully conserved residue among amino acid pemreases. According to a pKa prediction for E208 (pKa ∼8.3-11.7) of Escherichia coli AdiC antiporter, a structural homologue of Tat2, the E286 carboxyl chain of Tat2 could get loaded with a proton during tryptophan/H⁺ symport. Hence, I285V and I285T mutations might affect the buried residue environment of Tat2, thereby facilitating tryptophan import. Additionally, Tat2^I285V and Tat2^I285T levels increased rapidly, and were efficiently localized to the cell surface after transferring the cells to low tryptophan medium (0.5 μg/mL). Our findings provide a clue to gain insights into the property of high-affinity transport mechanisms, and offer a unique approach to improve the functionality of broad types of amino acid permeases.

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