Abstract
Chimeras of Hxt2 and Hxt1, high affinity and low affinity glucose transporters, respectively, of Saccharomyces cerevisiae, were previously constructed by random replacement of each of the 12 transmembrane segments (TMs) of Hxt2 with the corresponding region of Hxt1. Characterization of these chimeras revealed that at least TMs 1, 5, 7, and 8 of Hxt2 are required for high affinity transport activity. To determine which amino acid residues in these TMs are important for high affinity glucose transport, we systematically shuffled all of the 20 residues in these regions that differ between Hxt2 and Hxt1. Analysis of 60 independent mutant strains identified as expressing high affinity and high capacity glucose transport activity by selection on glucose-limited agar plates revealed that Leu-201 in TM5 of Hxt2 is most important for such activity and that either Cys-195 or Phe-198 is also required for maximal activity.
Highlights
Hexose transport across the plasma membrane is a necessary step in the utilization of monosaccharides by living cells
To determine which amino acid residues in these transmembrane segments (TMs) are important for high affinity glucose transport, we systematically shuffled all of the 20 residues in these regions that differ between Hxt2 and Hxt1
Analysis of 60 independent mutant strains identified as expressing high affinity and high capacity glucose transport activity by selection on glucose-limited agar plates revealed that Leu-201 in TM5 of Hxt2 is most important for such activity and that either Cys-195 or Phe-198 is required for maximal activity
Summary
Construction of Vectors—Construction of the plasmid Hxt2mnx-pVT, which comprises HXT2 under the control of the ADH1 promoter in the multicopy vector pVT102-U (YEp URA3 bla), was previously described [4]. In the first step, performed with ExTaq polymerase (Takara, Otsu, Japan), two DNA fragments that were designed to possess 7–10 overlapping nucleotides were prepared for mutants of each TM. These fragments encoded all the possible combinations of amino acid residues at the sites that differ between Hxt and Hxt. Plate Selection—Transformants that possessed high affinity, high capacity glucose transport activity were selected after incubation of yeast cells for 3 or 4 days at 30 °C on glucose-limited (glucose, 1 mg/ml) agar plates containing a synthetic medium supplemented with adenine.
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