Cysteine-scanning Mutagenesis of Transmembrane Segment 7 of the GLUT1 Glucose Transporter

The human erythrocyte facilitative glucose transporter (Glut1) is predicted to contain 12 transmembrane spanning α-helices based upon hydropathy plot analysis of the primary sequence. Five of these helices (3, 5, 7, 8, and 11) are capable of forming amphipathic structures. A model of GLUT1 tertiary structure has therefore been proposed in which the hydrophilic faces of several amphipathic helices are arranged to form a central aqueous channel through which glucose traverses the hydrophobic lipid bilayer. In order to test this model, we individually mutated each of the amino acid residues in transmembrane segment 7 to cysteine in an engineered GLUT1 molecule devoid of all native cysteines (C-less). Measurement of 2-deoxyglucose uptake in a Xenopus oocyte expression system revealed that nearly all of these mutants retain measurable transport activity. Over one-half of the cysteine mutants had significantly reduced specific activity relative to the C-less protein. The solvent accessibility and relative orientation of the residues within the helix was investigated by determining the sensitivity of the mutant transporters to inhibition by the sulfhydryl directed reagentp-chloromercuribenzene sulfonate (pCMBS). Cysteine replacement at six positions (Gln282, Gln283, Ile287, Ala289, Val290, and Phe291), all near the exofacial side of the cell membrane, produced transporters that were inhibited by incubation with extracellular pCMBS. Residues predicted to be near the cytoplasmic side of the cell membrane were minimally affected by pCMBS. These data demonstrate that the exofacial portion of transmembrane segment 7 is accessible to the external solvent and provide evidence for the positioning of this α-helix within the glucose permeation pathway. The human erythrocyte facilitative glucose transporter (Glut1) is predicted to contain 12 transmembrane spanning α-helices based upon hydropathy plot analysis of the primary sequence. Five of these helices (3, 5, 7, 8, and 11) are capable of forming amphipathic structures. A model of GLUT1 tertiary structure has therefore been proposed in which the hydrophilic faces of several amphipathic helices are arranged to form a central aqueous channel through which glucose traverses the hydrophobic lipid bilayer. In order to test this model, we individually mutated each of the amino acid residues in transmembrane segment 7 to cysteine in an engineered GLUT1 molecule devoid of all native cysteines (C-less). Measurement of 2-deoxyglucose uptake in a Xenopus oocyte expression system revealed that nearly all of these mutants retain measurable transport activity. Over one-half of the cysteine mutants had significantly reduced specific activity relative to the C-less protein. The solvent accessibility and relative orientation of the residues within the helix was investigated by determining the sensitivity of the mutant transporters to inhibition by the sulfhydryl directed reagentp-chloromercuribenzene sulfonate (pCMBS). Cysteine replacement at six positions (Gln282, Gln283, Ile287, Ala289, Val290, and Phe291), all near the exofacial side of the cell membrane, produced transporters that were inhibited by incubation with extracellular pCMBS. Residues predicted to be near the cytoplasmic side of the cell membrane were minimally affected by pCMBS. These data demonstrate that the exofacial portion of transmembrane segment 7 is accessible to the external solvent and provide evidence for the positioning of this α-helix within the glucose permeation pathway. p-chloromercuribenzene sulfonate Glut1 in which all 6 native cysteine residues were changed to either glycine or serine 2-deoxyglucose transmembrane plasma membrane The facilitative transport of glucose across mammalian cell membranes is mediated by a family of at least four highly homologous 50–60-kDa transmembrane glycoproteins (1Baldwin S.A. Biochim. Biophys. Acta. 1993; 1154: 17-49Crossref PubMed Scopus (279) Google Scholar, 2Mueckler M. Eur. J. Biochem. 1994; 219: 713-725Crossref PubMed Scopus (944) Google Scholar). Glut1, the prototype member of this family, has been the most extensively studied Glut protein because of its relative abundance within the erythrocyte cell membrane. It remains the only glucose transporter to have been purified and reconstituted into lipid vesicles in a functional form (3Kasahara M. Hinkle P.C. J. Biol. Chem. 1977; 252: 7384-7390Abstract Full Text PDF PubMed Google Scholar). The kinetics of glucose transport within the erythrocyte membrane has been extensively studied (4Carruthers A. Physiol. Rev. 1990; 70: 1135-1176Crossref PubMed Scopus (324) Google Scholar). Several amino acid residues that likely play crucial roles in glucose binding and/or transport have been identified from affinity labeling studies and limited site-directed mutagenesis (5Hashiramoto M. Kadowaki T. Clark A.E. Muraoka A. Momomura K. Sakura H. Tobe K. Akanuma Y. Yazaki Y. Holman G.D. Kasuga M. J. Biol. Chem. 1992; 267: 17502-17507Abstract Full Text PDF PubMed Google Scholar, 6Mueckler M. Weng W. Kruse M. J. Biol. Chem. 1994; 269: 20533-20538Abstract Full Text PDF PubMed Google Scholar, 7Garcia J.C. Strube M. Leingang K. Keller K. Mueckler M.M. J. Biol. Chem. 1992; 267: 7770-7776Abstract Full Text PDF PubMed Google Scholar). The tertiary structure of the Glut proteins, however, remains poorly characterized.A 12-transmembrane α-helical model for the glucose transporters was first proposed by Mueckler et al. (8Mueckler M. Caruso C. Baldwin S.A. Panico M. Blench I. Science. 1985; 229: 941-945Crossref PubMed Scopus (1131) Google Scholar) in 1985 based upon hydropathy plot analysis of the primary sequence. Fourier transform infrared spectroscopy and circular dichroism data determined that the protein is largely α-helical with the helices perpendicularly arranged relative to the lipid bilayer, in agreement with this model (9Alvarez J. Lee D.C. Baldwin S.A. Chapman D. J. Biol. Chem. 1987; 262: 3502-3509Abstract Full Text PDF PubMed Google Scholar, 10Chin J.J. Jung E.K. Chen V. Jung C.Y. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4113-4116Crossref PubMed Scopus (38) Google Scholar). This structure has been confirmed by enzymatic (11Cairns M.T. Alvarez J. Panico M. Gibbs A.F. Morris H.R. Chapman D. Baldwin S.A. Biochim. Biophys. Acta. 1987; 905: 295-310Crossref PubMed Scopus (61) Google Scholar), immunologic (12Davies A. Ciardelli T.L. Lienhard G.E. Boyle J.M. Whetton A.D. Baldwin S.A. Biochem. J. 1990; 266: 799-808PubMed Google Scholar), and glycosylation-scanning mutagenesis analysis (13Hresko R.C. Kruse M. Strube M. Mueckler M. J. Biol. Chem. 1994; 269: 20482-20488Abstract Full Text PDF PubMed Google Scholar). Hydrogen exchange experiments have revealed that 80% of the polypeptide backbone is solvent accessible, suggesting a pore-like structure (9Alvarez J. Lee D.C. Baldwin S.A. Chapman D. J. Biol. Chem. 1987; 262: 3502-3509Abstract Full Text PDF PubMed Google Scholar). Recognition that five of the putative transmembrane helices (numbers 3, 5, 7, 8, and 11) are capable of forming amphipathic helices has led to the hypothesis that these helices are arranged together to form an aqueous pathway that the glucose molecule traverses through the cell membrane (8Mueckler M. Caruso C. Baldwin S.A. Panico M. Blench I. Science. 1985; 229: 941-945Crossref PubMed Scopus (1131) Google Scholar). Molecular modeling of Glut1 has suggested that at least 5 transmembrane α-helices would be required to form an aqueous channel of sufficient size to accommodate the glucose moiety (14Zeng H. Parthasarathy R. Rampal A.L. Jung C.Y. Biophys. J. 1996; 70: 14-21Abstract Full Text PDF PubMed Scopus (45) Google Scholar).Support for the participation of TM segment 5 in forming part of this aqueous channel was recently obtained using cysteine-scanning mutagenesis. The sensitivity of Glut1 to mutations of Gln282 (5Hashiramoto M. Kadowaki T. Clark A.E. Muraoka A. Momomura K. Sakura H. Tobe K. Akanuma Y. Yazaki Y. Holman G.D. Kasuga M. J. Biol. Chem. 1992; 267: 17502-17507Abstract Full Text PDF PubMed Google Scholar) and Tyr293 (16Mori H. Hashiramoto M. Clark A.E. Yang J. Muraoka A. Tamori Y. Kasuga M. Holman G. J. Biol. Chem. 1994; 269: 11578-11583Abstract Full Text PDF PubMed Google Scholar) in TM segment 7 suggested that this helix may also comprise part of this glucose permeation pathway. In order to investigate the functional importance, solvent accessibility, and relative orientation of helix 7 with respect to this aqueous glucose channel, we initiated a systematic study of each TM amino acid by performing cysteine-scanning mutagenesis and measured the sensitivity of each of these mutants to modification by the sulfhydryl-directed reagent pCMBS. We report here evidence that the exofacial portion of TM segment 7 in GLUT1 contains solvent accessible residues that may be positioned within or near the glucose permeation pathway.DISCUSSIONHelical wheel analysis of the effects of cysteine-scanning mutagenesis and pCMBS modification in TM segment 7 contrasts with the results recently reported for TM segment 5 of GLUT1 (15Makepeace C. Mueckler M. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Analysis of the pCMBS inhibition pattern in helix 5 revealed that all of the residues sensitive to extracellular exposure to this reagent clustered along a single face of an α-helix. In TM segment 7, pCMBS-sensitive residues were positioned over a majority of the circumference of a helical wheel plot, rather than along a single face (Fig. 6). Olsowski and colleagues (24Olsowski A. Monden I. Keller K. Biochemistry. 1998; 37: 10738-10745Crossref PubMed Scopus (20) Google Scholar) also reported pCMBS sensitivity of residues within the exofacial portion of TM segment 7 in their study on the boundary between TM segment 7 and extracellular loop 4. In contrast to our results, Asn288was reported to be sensitive and Ala289 resistant to pCMBS modification. The specific activities of the cysteine mutants, however, were not determined in this study.The reduced specific activity observed in over half of the helix 7 mutants suggests an important structural role for this TM segment. However, it does not appear that any of the helix 7 amino acids are essential for sugar transport since nearly all of the mutants retain some residual activity. Although the mutation of Asn288 to cysteine resulted in a greater than 10-fold reduction in specific activity, mutation of this residue to Ile has been previously shown to have little effect on transport (5Hashiramoto M. Kadowaki T. Clark A.E. Muraoka A. Momomura K. Sakura H. Tobe K. Akanuma Y. Yazaki Y. Holman G.D. Kasuga M. J. Biol. Chem. 1992; 267: 17502-17507Abstract Full Text PDF PubMed Google Scholar). While pCMBS sensitivity clearly demonstrates that a portion of helix 7 is exposed to aqueous solvent and suggests positioning close to the glucose permeation pathway, helices 5 and 7 must have different orientations relative to the aqueous The data that the of helix 7 is highly accessible to suggesting that it within the putative aqueous have on the of the 12 transmembrane α-helices in Glut1 (1Baldwin S.A. Biochim. Biophys. Acta. 1993; 1154: 17-49Crossref PubMed Scopus (279) Google Scholar, Holman G.D. Biochem. J. 1993; PubMed Scopus Google Scholar). al. (14Zeng H. Parthasarathy R. Rampal A.L. Jung C.Y. Biophys. J. 1996; 70: 14-21Abstract Full Text PDF PubMed Scopus (45) Google Scholar) proposed for Glut1 helix that accommodate a model is with the positioning of helices 5 and 7 within the glucose permeation pathway. This model also that helices 8, and in forming the aqueous The of pCMBS-sensitive residues on of helix 7, however, is to with this specific helix most data the tertiary structure of the transport has been obtained for the from The of the 12 α-helices within this protein has been through a of cysteine-scanning and S. M. H.R. J. 1998; PubMed Scopus Google Scholar). 7 within the with to helices 5, and the of in membrane between of the transporter it is to that the glucose transporters a helix with the The positioning of helix 7 in Glut1 within the of the helix (Fig. is with pCMBS-sensitive residues on faces of the α-helix. It is that TM segment 7 has a of of the α-helix to be solvent accessible glucose of this helix by pCMBS modification on either side of the helix glucose model for Glut1 helix of a of the 12 transmembrane α-helices in from S. M. H.R. J. 1998; PubMed Scopus Google Scholar) model for helix within the to any pCMBS-sensitive residues within the cytoplasmic half of TM segment 7 is to the results obtained from cysteine-scanning mutagenesis of TM segment 5 (15Makepeace C. Mueckler M. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The inhibition pattern that either pCMBS is not accessible to the cytoplasmic portion of the α-helix from the external solvent or that modification is not sufficient to transport activity. The is with the model for the of glucose transport the cytoplasmic and exofacial are not accessible to solvent from the side of the lipid membrane. These results are also with the accessibility of the helix 7 TM amino acids in the of P.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google cysteine-scanning mutagenesis is to be a in the of the tertiary structure of membrane a of cysteine mutants for of the 12 in Glut1 it be to of the between these α-helices by performing experiments with of our study to the TM α-helices also likely provide into the of facilitative glucose This of the for Glut1 tertiary The facilitative transport of glucose across mammalian cell membranes is mediated by a family of at least four highly homologous 50–60-kDa transmembrane glycoproteins (1Baldwin S.A. Biochim. Biophys. Acta. 1993; 1154: 17-49Crossref PubMed Scopus (279) Google Scholar, 2Mueckler M. Eur. J. Biochem. 1994; 219: 713-725Crossref PubMed Scopus (944) Google Scholar). Glut1, the prototype member of this family, has been the most extensively studied Glut protein because of its relative abundance within the erythrocyte cell membrane. It remains the only glucose transporter to have been purified and reconstituted into lipid vesicles in a functional form (3Kasahara M. Hinkle P.C. J. Biol. Chem. 1977; 252: 7384-7390Abstract Full Text PDF PubMed Google Scholar). The kinetics of glucose transport within the erythrocyte membrane has been extensively studied (4Carruthers A. Physiol. Rev. 1990; 70: 1135-1176Crossref PubMed Scopus (324) Google Scholar). Several amino acid residues that likely play crucial roles in glucose binding and/or transport have been identified from affinity labeling studies and limited site-directed mutagenesis (5Hashiramoto M. Kadowaki T. Clark A.E. Muraoka A. Momomura K. Sakura H. Tobe K. Akanuma Y. Yazaki Y. Holman G.D. Kasuga M. J. Biol. Chem. 1992; 267: 17502-17507Abstract Full Text PDF PubMed Google Scholar, 6Mueckler M. Weng W. Kruse M. J. Biol. Chem. 1994; 269: 20533-20538Abstract Full Text PDF PubMed Google Scholar, 7Garcia J.C. Strube M. Leingang K. Keller K. Mueckler M.M. J. Biol. Chem. 1992; 267: 7770-7776Abstract Full Text PDF PubMed Google Scholar). The tertiary structure of the Glut proteins, however, remains poorly A 12-transmembrane α-helical model for the glucose transporters was first proposed by Mueckler et al. (8Mueckler M. Caruso C. Baldwin S.A. Panico M. Blench I. Science. 1985; 229: 941-945Crossref PubMed Scopus (1131) Google Scholar) in 1985 based upon hydropathy plot analysis of the primary sequence. Fourier transform infrared spectroscopy and circular dichroism data determined that the protein is largely α-helical with the helices perpendicularly arranged relative to the lipid bilayer, in agreement with this model (9Alvarez J. Lee D.C. Baldwin S.A. Chapman D. J. Biol. Chem. 1987; 262: 3502-3509Abstract Full Text PDF PubMed Google Scholar, 10Chin J.J. Jung E.K. Chen V. Jung C.Y. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4113-4116Crossref PubMed Scopus (38) Google Scholar). This structure has been confirmed by enzymatic (11Cairns M.T. Alvarez J. Panico M. Gibbs A.F. Morris H.R. Chapman D. Baldwin S.A. Biochim. Biophys. Acta. 1987; 905: 295-310Crossref PubMed Scopus (61) Google Scholar), immunologic (12Davies A. Ciardelli T.L. Lienhard G.E. Boyle J.M. Whetton A.D. Baldwin S.A. Biochem. J. 1990; 266: 799-808PubMed Google Scholar), and glycosylation-scanning mutagenesis analysis (13Hresko R.C. Kruse M. Strube M. Mueckler M. J. Biol. Chem. 1994; 269: 20482-20488Abstract Full Text PDF PubMed Google Scholar). Hydrogen exchange experiments have revealed that 80% of the polypeptide backbone is solvent accessible, suggesting a pore-like structure (9Alvarez J. Lee D.C. Baldwin S.A. Chapman D. J. Biol. Chem. 1987; 262: 3502-3509Abstract Full Text PDF PubMed Google Scholar). Recognition that five of the putative transmembrane helices (numbers 3, 5, 7, 8, and 11) are capable of forming amphipathic helices has led to the hypothesis that these helices are arranged together to form an aqueous pathway that the glucose molecule traverses through the cell membrane (8Mueckler M. Caruso C. Baldwin S.A. Panico M. Blench I. Science. 1985; 229: 941-945Crossref PubMed Scopus (1131) Google Scholar). Molecular modeling of Glut1 has suggested that at least 5 transmembrane α-helices would be required to form an aqueous channel of sufficient size to accommodate the glucose moiety (14Zeng H. Parthasarathy R. Rampal A.L. Jung C.Y. Biophys. J. 1996; 70: 14-21Abstract Full Text PDF PubMed Scopus (45) Google Scholar). for the participation of TM segment 5 in forming part of this aqueous channel was recently obtained using cysteine-scanning mutagenesis. The sensitivity of Glut1 to mutations of Gln282 (5Hashiramoto M. Kadowaki T. Clark A.E. Muraoka A. Momomura K. Sakura H. Tobe K. Akanuma Y. Yazaki Y. Holman G.D. Kasuga M. J. Biol. Chem. 1992; 267: 17502-17507Abstract Full Text PDF PubMed Google Scholar) and Tyr293 (16Mori H. Hashiramoto M. Clark A.E. Yang J. Muraoka A. Tamori Y. Kasuga M. Holman G. J. Biol. Chem. 1994; 269: 11578-11583Abstract Full Text PDF PubMed Google Scholar) in TM segment 7 suggested that this helix may also comprise part of this glucose permeation pathway. In order to investigate the functional importance, solvent accessibility, and relative orientation of helix 7 with respect to this aqueous glucose channel, we initiated a systematic study of each TM amino acid by performing cysteine-scanning mutagenesis and measured the sensitivity of each of these mutants to modification by the sulfhydryl-directed reagent pCMBS. We report here evidence that the exofacial portion of TM segment 7 in GLUT1 contains solvent accessible residues that may be positioned within or near the glucose permeation pathway. wheel analysis of the effects of cysteine-scanning mutagenesis and pCMBS modification in TM segment 7 contrasts with the results recently reported for TM segment 5 of GLUT1 (15Makepeace C. Mueckler M. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Analysis of the pCMBS inhibition pattern in helix 5 revealed that all of the residues sensitive to extracellular exposure to this reagent clustered along a single face of an α-helix. In TM segment 7, pCMBS-sensitive residues were positioned over a majority of the circumference of a helical wheel plot, rather than along a single face (Fig. 6). Olsowski and colleagues (24Olsowski A. Monden I. Keller K. Biochemistry. 1998; 37: 10738-10745Crossref PubMed Scopus (20) Google Scholar) also reported pCMBS sensitivity of residues within the exofacial portion of TM segment 7 in their study on the boundary between TM segment 7 and extracellular loop 4. In contrast to our results, Asn288was reported to be sensitive and Ala289 resistant to pCMBS modification. The specific activities of the cysteine mutants, however, were not determined in this study.The reduced specific activity observed in over half of the helix 7 mutants suggests an important structural role for this TM segment. However, it does not appear that any of the helix 7 amino acids are essential for sugar transport since nearly all of the mutants retain some residual activity. Although the mutation of Asn288 to cysteine resulted in a greater than 10-fold reduction in specific activity, mutation of this residue to Ile has been previously shown to have little effect on transport (5Hashiramoto M. Kadowaki T. Clark A.E. Muraoka A. Momomura K. Sakura H. Tobe K. Akanuma Y. Yazaki Y. Holman G.D. Kasuga M. J. Biol. Chem. 1992; 267: 17502-17507Abstract Full Text PDF PubMed Google Scholar). While pCMBS sensitivity clearly demonstrates that a portion of helix 7 is exposed to aqueous solvent and suggests positioning close to the glucose permeation pathway, helices 5 and 7 must have different orientations relative to the aqueous The data that the of helix 7 is highly accessible to suggesting that it within the putative aqueous have on the of the 12 transmembrane α-helices in Glut1 (1Baldwin S.A. Biochim. Biophys. Acta. 1993; 1154: 17-49Crossref PubMed Scopus (279) Google Scholar, Holman G.D. Biochem. J. 1993; PubMed Scopus Google Scholar). al. (14Zeng H. Parthasarathy R. Rampal A.L. Jung C.Y. Biophys. J. 1996; 70: 14-21Abstract Full Text PDF PubMed Scopus (45) Google Scholar) proposed for Glut1 helix that accommodate a model is with the positioning of helices 5 and 7 within the glucose permeation pathway. This model also that helices 8, and in forming the aqueous The of pCMBS-sensitive residues on of helix 7, however, is to with this specific helix most data the tertiary structure of the transport has been obtained for the from The of the 12 α-helices within this protein has been through a of cysteine-scanning and S. M. H.R. J. 1998; PubMed Scopus Google Scholar). 7 within the with to helices 5, and the of in membrane between of the transporter it is to that the glucose transporters a helix with the The positioning of helix 7 in Glut1 within the of the helix (Fig. is with pCMBS-sensitive residues on faces of the α-helix. It is that TM segment 7 has a of of the α-helix to be solvent accessible glucose of this helix by pCMBS modification on either side of the helix glucose to any pCMBS-sensitive residues within the cytoplasmic half of TM segment 7 is to the results obtained from cysteine-scanning mutagenesis of TM segment 5 (15Makepeace C. Mueckler M. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The inhibition pattern that either pCMBS is not accessible to the cytoplasmic portion of the α-helix from the external solvent or that modification is not sufficient to transport activity. The is with the model for the of glucose transport the cytoplasmic and exofacial are not accessible to solvent from the side of the lipid membrane. These results are also with the accessibility of the helix 7 TM amino acids in the of P.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google cysteine-scanning mutagenesis is to be a in the of the tertiary structure of membrane a of cysteine mutants for of the 12 in Glut1 it be to of the between these α-helices by performing experiments with of our study to the TM α-helices also likely provide into the of facilitative glucose This of the for Glut1 tertiary wheel analysis of the effects of cysteine-scanning mutagenesis and pCMBS modification in TM segment 7 contrasts with the results recently reported for TM segment 5 of GLUT1 (15Makepeace C. Mueckler M. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Analysis of the pCMBS inhibition pattern in helix 5 revealed that all of the residues sensitive to extracellular exposure to this reagent clustered along a single face of an α-helix. In TM segment 7, pCMBS-sensitive residues were positioned over a majority of the circumference of a helical wheel plot, rather than along a single face (Fig. 6). Olsowski and colleagues (24Olsowski A. Monden I. Keller K. Biochemistry. 1998; 37: 10738-10745Crossref PubMed Scopus (20) Google Scholar) also reported pCMBS sensitivity of residues within the exofacial portion of TM segment 7 in their study on the boundary between TM segment 7 and extracellular loop 4. In contrast to our results, Asn288was reported to be sensitive and Ala289 resistant to pCMBS modification. The specific activities of the cysteine mutants, however, were not determined in this The reduced specific activity observed in over half of the helix 7 mutants suggests an important structural role for this TM segment. However, it does not appear that any of the helix 7 amino acids are essential for sugar transport since nearly all of the mutants retain some residual activity. Although the mutation of Asn288 to cysteine resulted in a greater than 10-fold reduction in specific activity, mutation of this residue to Ile has been previously shown to have little effect on transport (5Hashiramoto M. Kadowaki T. Clark A.E. Muraoka A. Momomura K. Sakura H. Tobe K. Akanuma Y. Yazaki Y. Holman G.D. Kasuga M. J. Biol. Chem. 1992; 267: 17502-17507Abstract Full Text PDF PubMed Google Scholar). While pCMBS sensitivity clearly demonstrates that a portion of helix 7 is exposed to aqueous solvent and suggests positioning close to the glucose permeation pathway, helices 5 and 7 must have different orientations relative to the aqueous The data that the of helix 7 is highly accessible to suggesting that it within the putative aqueous Several have on the of the 12 transmembrane α-helices in Glut1 (1Baldwin S.A. Biochim. Biophys. Acta. 1993; 1154: 17-49Crossref PubMed Scopus (279) Google Scholar, Holman G.D. Biochem. J. 1993; PubMed Scopus Google Scholar). al. (14Zeng H. Parthasarathy R. Rampal A.L. Jung C.Y. Biophys. J. 1996; 70: 14-21Abstract Full Text PDF PubMed Scopus (45) Google Scholar) proposed for Glut1 helix that accommodate a model is with the positioning of helices 5 and 7 within the glucose permeation pathway. This model also that helices 8, and in forming the aqueous The of pCMBS-sensitive residues on of helix 7, however, is to with this specific helix The most data the tertiary structure of the transport has been obtained for the from The of the 12 α-helices within this protein has been through a of cysteine-scanning and S. M. H.R. J. 1998; PubMed Scopus Google Scholar). 7 within the with to helices 5, and the of in membrane between of the transporter it is to that the glucose transporters a helix with the The positioning of helix 7 in Glut1 within the of the helix (Fig. is with pCMBS-sensitive residues on faces of the α-helix. It is that TM segment 7 has a of of the α-helix to be solvent accessible glucose of this helix by pCMBS modification on either side of the helix glucose The to any pCMBS-sensitive residues within the cytoplasmic half of TM segment 7 is to the results obtained from cysteine-scanning mutagenesis of TM segment 5 (15Makepeace C. Mueckler M. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The inhibition pattern that either pCMBS is not accessible to the cytoplasmic portion of the α-helix from the external solvent or that modification is not sufficient to transport activity. The is with the model for the of glucose transport the cytoplasmic and exofacial are not accessible to solvent from the side of the lipid membrane. These results are also with the accessibility of the helix 7 TM amino acids in the of P.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Although cysteine-scanning mutagenesis is to be a in the of the tertiary structure of membrane a of cysteine mutants for of the 12 in Glut1 it be to of the between these α-helices by performing experiments with of our study to the TM α-helices also likely provide into the of facilitative glucose This of the for Glut1 tertiary