Identification by comprehensive chimeric analysis of a key residue responsible for high affinity glucose transport by yeast HXT2

Abstract

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 Leu61 (TM1), Leu201 (TM5), Asn 331 (TM7), and Phe366 (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 Asn331 yielded transporters with various affinities, with those of the Ile331, Val 331, and Cys331 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 Asn331 or its 19 replacement residues indicated that those residues at this site that yield high affinity transporters (Ile331, Val 331, Cys331) face the central cavity and are within van der Waals distances of Phe208 (TM5), Leu357 (TM8), and Tyr427 (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. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.