Article

Transmembrane segment 12 of the Glut1 glucose transporter is an outer helix and is not directly involved in the transport mechanism

Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 01/2007; 281(48):36993-8. DOI: 10.1074/jbc.M608158200
Source: PubMed

ABSTRACT A model has been proposed for the exofacial configuration of the Glut1 glucose transporter in which eight transmembrane domains form an inner helical bundle stabilized by four outer helices. The role of transmembrane segment 12, predicted to be an outer helix in this hypothetical model, was examined by cysteine-scanning mutagenesis and the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A previously characterized functional cysteine-less Glut1 molecule was used to produce 21 Glut1 point mutants by changing each residue along helix 12 to a cysteine residue. These mutants were then expressed in Xenopus oocytes, and their protein levels, functional activities, and sensitivities to pCMBS were determined. Strikingly, in contrast to all nine other predicted Glut1 transmembrane helices that have been previously examined by this method, none of the 21 helix 12 single-cysteine mutants exhibited significant inhibition of specific transport activity. Also unlike most other Glut1 transmembrane domains in which solvent-accessible residues lie along a single face of the helix, mutations in five consecutive residues predicted to lie close to the exofacial face of the membrane resulted in sensitivity to pCMBS-induced transport inhibition. These results suggest that helix 12 plays a passive stabilizing role in the structure of Glut1 and is not directly involved in the transport mechanism. Additionally, the pCMBS data indicate that the predicted exoplasmic end of helix 12 is completely exposed to the external solvent when the transporter is in its exofacial configuration.

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    • "Cysteine-scanning mutagenesis and substituted cysteine accessibility studies implicate transmembrane segments 1 [21], 2 [22], 5 [23], 7 [22], [24], 8 [25], 10 [26], and 11 [27] of Glut1 in the formation of a water-accessible cleft within the membrane. In contrast, helices 3 [28], 6 [29], 9 [30], and 12 [31] appear to have limited access to the external solvent, suggesting that these segments form the outer stabilizing helices as indicated by the known bacterial MFS structures [15], [16], [17], [18]. Transmembrane segment 4 of Glut1 does not appear to react with pCMBS added to the external solvent [32]. "
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    • "These studies revealed that transmembrane segments 1, 2, 4, 5, 7, 8, 10 and 11 form the glucose transport channel and its amphipathic nature suggests the possibility of an aqueous permeation pore for the transport of glucose [23]–[30]. The outer helices 3, 6, 9 and 12 stabilize the central channel [31]–[34]. Several amino acid residues important for the function of glucose transporters have been identified from mutagenesis studies, and many of these residues are conserved among the GLUT members. Noticeably, ATP is shown to regulate the GLUT1-mediated glucose transport activity, but it does not require any ATP hydrolysis [35]–[37]. "
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    • "It was deduced from these structures that TMS 3, 6, 9, and 12 are not involved in substrate binding or translocation, but—as scaffolding or pillars—encircle and stabilize the inner bundle of the other helices. This notion was fully supported by cysteine-scanning mutagenesis and cysteine reagent accessibility assays of TMS 9 and 12 of GLUT1 [28] [29]. However, since our mutant e5.7.10 with about 15% carnitine transport relative to wild-type was still far off the 50% mark, and since our evolutionary analysis indicated interesting positions in TMS 6, 9, and 12, they were included in the second set of ETTh mutants. "
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