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

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, 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...

Analysis of Glucose Transporter Topology and Structural Dynamics

“…153 and Lys 183 (this study), the unexpected accessibility of mutants Q161C and I164C of TM5 to interstitial 4-(chloromercuri)benzosulfonic acid (10), and the SDS-dependent accessibility of Arg 153 and Lys 183 (this study). TM12 adjustment to relocate Lys 451 one additional helix turn into the lipid bilayer (direction cytoplasm to interstitium) would explain the trypsin inaccessibility of Lys 451 (this study) and, by displacing N-terminal TM12 sequence into the interstitium, may also explain the unexpected accessibility of TM12 Y432C through I436C to interstitial 4-(chloromercuri)benzosulfonic acid (10). These interpretations must be tempered by the possibility of residue occlusion by local structure.…”

“…Homology modeling suggests involvement of GLUT1 TMs 2, 4, 5, 7, 8, and 10 in substrate binding (11), although caveats with this approach have been noted (22). Scanning cysteine mutagenesis implicates GLUT1 TMs 1, 5, 7, 8, and 11 in sugar binding (10). (19), which form the cytoplasmic loop and TM segments of the TM10-TM11 hairpin (11).…”

“…GLUT1 cysteinescanning mutagenesis studies (10) broadly support the resulting GLUT1 MFS ␣-helical packing arrangement but also note significant accessibility in TM regions that, according to the model, should be inaccessible (10). Docking analysis of cytochalasin B (a transport inhibitor) binding to homology-modeled GLUT1 positions the binding site within the cytoplasmic loop linking TMs 2 and 3 (11), whereas biochemical studies suggest that cytochalasin B binds close to cytoplasmic loop 10 -11 (19 -21).…”

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

“…45,62,63 Cysteine scanning mutagenesis was subsequently employed to probe which residues lined this proposed central pore. [64][65][66][67][68][69][70][71][72][73][74] The resulting model indicated that TMs 2, 4, 5, 7, 8, 11, and possibly 1 and 10 formed a central aqueous transport channel for glucose, whereas TMs 3, 6, 9, and 12 formed a structural scaffold on the outside of the protein. 70 A possible substrate binding site was also proposed, involving Q161, Q282, and W412.…”

The expression and regulation of glucose transporters in tumor cells

“…(online first, page number not for citation purpose) crease in the expression of glucose transporter in lung cancer and thymic epithelial tumors [18,29] . A higher expression of SGLT1 has also been found in ovarian, colorectal, and lung cancer tissues compared to healthy tissues, indicating a positive correlation between the activity of glucose transporters and tumor development [30] .…”