Journal of Biological Chemistry
Volume 285, Issue 34, 20 August 2010, Pages 26263-26268
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Membrane Biology
Identification of a Key Residue Determining Substrate Affinity in the Yeast Glucose Transporter Hxt7: A TWO-DIMENSIONAL COMPREHENSIVE STUDY*

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We previously identified Asn331 in transmembrane segment 7 (TM7) as a key residue determining substrate affinity in Hxt2, a moderately high-affinity facilitative glucose transporter of Saccharomyces cerevisiae. To gain further insight into the structural basis of substrate recognition by yeast glucose transporters, we have now studied Hxt7, whose affinity for glucose is the highest among the major hexose transporters. The functional role of Asp340 in Hxt7, the residue corresponding to Asn331 of Hxt2, was examined by replacing it with each of the other 19 amino acids. Such replacement of Asp340 generated transporters with various affinities for glucose, with the affinity of the Cys340 mutant surpassing that of the wild-type Hxt7. To examine the structural role of Asp340 in the substrate translocation pathway, we performed cysteine-scanning mutagenesis of the 21 residues in TM7 of a functional Cys-less Hxt7 mutant in conjunction with exposure to the hydrophilic sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMBS). The transport activity of the D340C mutant of Cys-less Hxt7, in which Asp340 is replaced with Cys, was completely inhibited by pCMBS, indicating that Asp340 is located in a water-accessible position. This D340C mutant showed a sensitivity to pCMBS that was ∼70 times that of the wild-type Hxt7, and it was protected from pCMBS inhibition by the substrates d-glucose and 2-deoxy-d-glucose but not by l-glucose. These results indicate that Asp340 is situated at or close to a substrate recognition site and is a key residue determining high-affinity glucose transport by Hxt7, supporting the notion that yeast glucose transporters share a common mechanism for substrate recognition.

Aspartate
Glucose Transport
Membrane Function
Membrane Proteins
Sugar Transport
Yeast
Cysteine-scanning Mutagenesis
Membrane Transport
Substrate Recognition

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*

This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and from Teikyo University.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2 and Figs. S1–S4.