Transmembrane Segment 3 of the Glut1 Glucose Transporter Is an Outer Helix

2006

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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, sulfhydrylspecific 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.The transport of glucose across mammalian cell membranes is mediated by proteins produced by the Glut gene family (reviewed in Refs. 1-3), part of the major facilitator superfamily (MFS) 2 that contains several thousand members, present in virtually every organism examined thus far (4). MFS proteins are involved in the transport of a diverse collection of low molecular weight molecules across membranes (5).Glut1 is expressed at its highest known levels in the human erythrocyte and is the only member of the Glut protein family to have been purified (6, 7). It remains one of the most extensively studied and thoroughly characterized members of the MFS (8). Based on analysis of the protein sequence deduced from a human cDNA clone, Glut1 was the first MFS protein predicted to possess 12 transmembrane helices (9), a major feature of most if not all members of this superfamily. A number of experimental observations, most notably a comprehensive glycosylation-scanning mutagenesis study (10), are consistent with this structural prediction. Several of the 12 predicted transmembrane helices are amphipathic, an observation that led to the suggestion that these helices form an exposed cavity in the membrane-buried portion of the protein that participates in the binding and translocation of glucose across the lipid bilayer (9). Hydrogen bond-forming amino acid side chains within the...

supporting

confidence: 73%

Transmembrane Segment 12 of the Glut1 Glucose Transporter Is an Outer Helix and Is Not Directly Involved in the Transport Mechanism

2006

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“…Helices 3, 6, 9, and 12 are predicted to surround this inner helical bundle. Our recently reported analysis of helix 3 is consistent with it being an outer helix (18).…”

supporting

confidence: 66%

Cysteine-scanning Mutagenesis and Substituted Cysteine Accessibility Analysis of Transmembrane Segment 4 of the Glut1 Glucose Transporter

2005

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A low resolution model has been proposed for the exofacial conformation of the Glut1 glucose transporter in which eight transmembrane segments form an inner helical bundle stabilized by four outer helices. The role of transmembrane segment 4, predicted to be an inner helix in this structural model, was investigated by cysteinescanning mutagenesis in conjunction with the substituted cysteine accessibility method using the membrane-impermeant, sulfhydrylspecific reagent, p-chloromercuribenzenesulfonate (pCMBS). A functional, cysteine-less, parental Glut1 molecule was used to produce 21 Glut1 point mutants by individually changing each residue along transmembrane helix 4 to a cysteine. The single cysteine mutants were then expressed in Xenopus oocytes, and their expression levels, transport activities, and sensitivities to pCMBS were determined. In striking contrast to all of the other seven predicted inner helices, none of the 21 helix 4 single-cysteine mutants was demonstrably inhibited by pCMBS. However, cysteine substitution within helix 4 resulted in an unusually high number of severely transport-defective mutants. The low absolute transport activities of two of these mutants (G130C and G134C) were due to their extremely low levels of expression, presumably a result of structural instability and consequent degradation in oocytes, suggesting that these two residues play an important role in maintaining the native structure of Glut1. The other two transport-defective mutants (Y143C and E146C) exhibited low specific transport activities, implying that these two residues play an important role in the transport cycle. Based on these data, we conclude that the exoplasmic end of helix 4 lies outside the inner helical bundle in the exofacial configuration of Glut1.

“…Data suggest that transmembrane segments 3 and 12 are outer helices that stabilize the inner bundle (20,21). This experimentally derived model is largely consistent with homology modeling of Glut1 based on the high resolution structures recently described for the lac permease (22) and glycerol-3-P antiporter (23), two bacterial members of the MFS expressed in Escherichia coli.…”

supporting

confidence: 66%

Transmembrane Segment 6 of the Glut1 Glucose Transporter Is an Outer Helix and Contains Amino Acid Side Chains Essential for Transport Activity

2008

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Experimental data and homology modeling suggest a structure for the exofacial configuration of the Glut1 glucose transporter in which 8 transmembrane helices form an aqueous cavity in the bilayer that is stabilized by four outer helices. The role of transmembrane segment 6, predicted to be an outer helix in this model, was examined by cysteine-scanning mutagenesis and the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzene-sulfonate (pCMBS). A fully functional Glut1 molecule lacking all 6 native cysteine residues was used as a template to produce a series of 21 Glut1 point mutants in which each residue along helix 6 was individually changed to cysteine. These mutants were expressed in Xenopus oocytes, and their expression levels, functional activities, and sensitivities to inhibition by pCMBS were determined. moderately augmented specific transport activity relative to the control. These results were dramatically different from those previously reported for helix 12, the structural cognate of helix 6 in the pseudo-symmetrical structural model, for which none of the 21 single-cysteine mutants exhibited reduced activity. Only the substitution at Leu 188 conferred inhibition by pCMBS, suggesting that most of helix 6 is not exposed to the external solvent, consistent with its proposed role as an outer helix. These data suggest that helix 6 contains amino acid side chains that are critical for transport activity and that structurally analogous outer helices may play distinct roles in the function of membrane transporters.