Comprehensive chimeric analysis of amino acid residues critical for high affinity glucose transport by Hxt2 of Saccharomyces cerevisiae.
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.
Kasahara
Toshiko
T
Laboratory of Biophysics, School of Medicine, Teikyo University, Hachioji, Tokyo 192-0395, Japan. kasahara@main.teikyo-u.ac.jp
Ishiguro
Masaji
M
Kasahara
Michihiro
M
D000596:Amino Acids / Q000737:chemistry*; D001692:Biological Transport; D003545:Cysteine / Q000737:chemistry; D005822:Genetic Vectors; D005947:Glucose / Q000737:chemistry / Q000378:metabolism*; D051246:Glucose Transport Proteins, Facilitative; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*
D000596:Amino Acids; D051246:Glucose Transport Proteins, Facilitative; C090594:HXT1 protein, S cerevisiae; C084498:HXT2 protein, S cerevisiae; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D010649:Phenylalanine; D007930:Leucine; D005947:Glucose; D003545:Cysteine