Identification of a Hydrophobic Residue as a Key Determinant of Fructose Transport by the Facilitative Hexose Transporter SLC2A7 (GLUT7)

Abstract

Until recently, the only facilitated hexose transporter GLUT proteins (SLC2A) known to transport fructose were GLUTs 2 and 5. However, the recently cloned GLUT7 can also transport fructose as well as glucose. Comparison of sequence alignments indicated that GLUTs 2, 5, and 7 all had an isoleucine residue at position "314" (GLUT7), whereas the non-fructose-transporting isoforms, GLUTs 1, 3, and 4, had a valine at this position. Mutation of Ile-314 to a valine in GLUT7 resulted in a loss of fructose transport, whereas glucose transport remained completely unaffected. Similar results were obtained with GLUTs 2 and 5. Energy minimization modeling of GLUT7 indicated that Ile-314 projects from transmembrane domain 7 (TM7) into the lumen of the aqueous pore, where it could form a hydrophobic interaction with tryptophan 89 from TM2. A valine residue at 314 appeared to produce a narrowing of the vestibule when compared with the isoleucine. It is proposed that this hydrophobic interaction across the pore forms a selectivity filter restricting the access of some hexoses to the substrate binding site(s) within the aqueous channel. The presence of a selectivity filter in the extracellular vestibule of GLUT proteins would allow for subtle changes in substrate specificity without changing the kinetic parameters of the protein.The facilitative glucose transporters (SLC2a) belong to the facilitated transporter super gene family, which all appear to have a core structure of 12 transmembrane helices clustered in two sets of six between which there is a central aqueous pore (1). The recent crystal structures solved for LacY and GlpT suggest that during the transport cycle, these two clusters undergo an alteration in their tilt such that their binding site moves from an outward-to an inward-facing conformation (2, 3). In the case of the GLUT 3 proteins, scanning mutagenesis studies and computer modeling indicate that their pore is formed by TMs 5, 7, 8, 10, and 11, whereas other helices may influence the pore structure, i.e. TMs 1, 2, 3, and 4 (4 -6). What is less clear is the shape of the outer-or innerfacing vestibules allowing entry of the substrates into the pore and access to the proposed substrate binding site. Mueckler et al. (7) initially reported that a naturally occurring conservative mutation of valine 197 to isoleucine (V197I) in GLUT2 resulted in a greatly reduced transport capacity when the protein was expressed in Xenopus oocytes. Subsequently, they also reported that a comparable mutation in GLUT1 (valine 165 to isoleucine) abolished glucose transport (8). Valine is a smaller residue than isoleucine, and this raised the possibility that, if these residues in TM5 faced into the pore, the larger isoleucine might hinder the passage of substrate. These results did not explain why such a conservative substitution could have such a profound effect on the function of these proteins. However, it has been proposed that one or more hydrophobic residues projecting into the pore can influence substrate access to the binding sit...

Identification by Comprehensive Chimeric Analysis of a Key Residue Responsible for High Affinity Glucose Transport by Yeast HXT2

“…Exposure of cells expressing a single Cys-substituted form of GLUT1 to a water-soluble sulfhydryl reagent revealed that the exofacial portion of TM7 is accessible to the external solvent (19). A QLS motif present in TM7 of GLUT family proteins has been proposed to interact with the C-1 position of D-glucose (20), and Ile 314 situated at the outer edge of TM7 of GLUT7 was shown to be required for fructose transport (21). Studies of other MFS transporters have also shown the importance of TM7.…”

Fructose transporter Glut5 expression in clear renal cell carcinoma

“…Fructose is transported passively across membranes by a member of the facilitative glucose transporter (GLUT) family, named GLUT5 (60)(61)(62)(63)(64) and it is the sole transporter specific for fructose with no ability to transport glucose or galactose. The low intracellular fructose concentration is possible because fructose is metabolized and significantly contributes to glycogenolysis in muscle or lipogenesis in adipocytes (63,65).…”

Structure of, and functional insight into the GLUT family of membrane transporters

“…59 It was speculated that this residue was facing the aqueous pore at the exofacial vestibule of GLUT7 using a computer model of the GLUT7 protein based on the GlpT crystal structure. 60 However, analysis using a GLUT9 computer model based on the recent GLUT1 crystal structure indicates that this isoleucine is more likely to be facing away from the aqueous pore and to be acting as a structural regulator through hydrophobic interactions with adjacent TMs. 61 At the equivalent position in Class I GLUTs (except for GLUT2 which can also transport fructose) there is a valine residue.…”