Identification by comprehensive chimeric analysis of a key residue responsible for high affinity glucose transport by yeast HXT2.
Hxt2 and Hxt1 are, respectively, high affinity and low affinity facilitative glucose transporter paralogs of Saccharomyces cerevisiae. We have previously investigated which amino acid residues of Hxt2 are important for high affinity transport activity. Studies with all the possible combinations of 12 transmembrane segments (TMs) of Hxt2 and Hxt1 revealed that TMs 1, 5, 7, and 8 of Hxt2 are necessary for high affinity transport. Systematic shuffling of the 20 amino acid residues that differ between Hxt2 and Hxt1 in these TMs subsequently identified 5 residues as important for such activity: Leu(59) and Leu(61) (TM1), Leu(201) (TM5), Asn(331) (TM7), and Phe(366) (TM8). We have now studied the relative importance of these 5 residues by individually replacing them with each of the other 19 residues. Replacement of Asn(331) yielded transporters with various affinities, with those of the Ile(331), Val(331), and Cys(331) mutants being higher than that of the wild type. Replacement of the Hxt2 residues at the other four sites yielded transporters with affinities similar to that of the wild type but with various capacities. A working homology model of the chimeric transporters containing Asn(331) or its 19 replacement residues indicated that those residues at this site that yield high affinity transporters (Ile(331), Val(331), Cys(331)) face the central cavity and are within van der Waals distances of Phe(208) (TM5), Leu(357) (TM8), and Tyr(427) (TM10). Interactions via these residues of the four TMs, which compose a half of the central pore, may thus play a pivotal role in formation of a core structure for high affinity transport.
Kasahara
Toshiko
T
Laboratory of Biophysics, School of Medicine, Teikyo University, Hachioji, Tokyo 192-0395.
Maeda
Mari
M
Ishiguro
Masaji
M
Kasahara
Michihiro
M
D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism; D001693:Biological Transport, Active / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D051246:Glucose Transport Proteins, Facilitative; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000235:genetics* / Q000378:metabolism; D050506:Mutant Chimeric Proteins / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
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; D050506:Mutant Chimeric Proteins; D029701:Saccharomyces cerevisiae Proteins; D005947:Glucose