Analysis of Transmembrane Segment 8 of the GLUT1 Glucose Transporter by Cysteine-scanning Mutagenesis and Substituted Cysteine Accessibility
2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês
10.1074/jbc.m310786200
ISSN1083-351X
AutoresMike Mueckler, Carol M. Makepeace,
Tópico(s)Enzyme Structure and Function
ResumoThe GLUT1 glucose transporter has been proposed to form an aqueous substrate translocation pathway via the clustering of several amphipathic transmembrane helices (Mueckler, M., Caruso, C., Baldwin, S. A., Panico, M., Blench, I., Morris, H. R., Allard, W. J., Lienhard, G. E., and Lodish, H. F. (1985) Science 229, 941–945). The possible role of transmembrane helix 8 in the formation of this permeation pathway was investigated using cysteine-scanning mutagenesis and the membrane-impermeant sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). Twenty-one GLUT1 mutants were created from a fully functional cysteine-less parental GLUT1 molecule by successively changing each residue along transmembrane segment 8 to a cysteine. The mutant proteins were then expressed in Xenopus oocytes, and their membrane concentrations, 2-deoxyglucose uptake activities, and sensitivities to pCMBS were determined. Four positions within helix 8, alanine 309, threonine 310, serine 313, and glycine 314, were accessible to pCMBS as judged by the inhibition of transport activity. All four of these residues are clustered along one face of a putative α-helix. These results suggest that transmembrane segment 8 of GLUT1 forms part of the sugar permeation pathway. Updated two-dimensional models for the orientation of the 12 transmembrane helices and the conformation of the exofacial glucose binding site of GLUT1 are proposed that are consistent with existing experimental data and homology modeling based on the crystal structures of two bacterial membrane transporters. The GLUT1 glucose transporter has been proposed to form an aqueous substrate translocation pathway via the clustering of several amphipathic transmembrane helices (Mueckler, M., Caruso, C., Baldwin, S. A., Panico, M., Blench, I., Morris, H. R., Allard, W. J., Lienhard, G. E., and Lodish, H. F. (1985) Science 229, 941–945). The possible role of transmembrane helix 8 in the formation of this permeation pathway was investigated using cysteine-scanning mutagenesis and the membrane-impermeant sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). Twenty-one GLUT1 mutants were created from a fully functional cysteine-less parental GLUT1 molecule by successively changing each residue along transmembrane segment 8 to a cysteine. The mutant proteins were then expressed in Xenopus oocytes, and their membrane concentrations, 2-deoxyglucose uptake activities, and sensitivities to pCMBS were determined. Four positions within helix 8, alanine 309, threonine 310, serine 313, and glycine 314, were accessible to pCMBS as judged by the inhibition of transport activity. All four of these residues are clustered along one face of a putative α-helix. These results suggest that transmembrane segment 8 of GLUT1 forms part of the sugar permeation pathway. Updated two-dimensional models for the orientation of the 12 transmembrane helices and the conformation of the exofacial glucose binding site of GLUT1 are proposed that are consistent with existing experimental data and homology modeling based on the crystal structures of two bacterial membrane transporters. Passive transport of glucose across the plasma membrane of mammalian cells is mediated by members of the GLUT (SLC2a) family of membrane glycoproteins (reviewed in Refs. 1Baldwin S.A. Biochim. Biophys. Acta. 1993; 1154: 17-49Crossref PubMed Scopus (280) Google Scholar, 2Mueckler M. Eur. J. Biochem. 1994; 219: 713-725Crossref PubMed Scopus (958) Google Scholar, 3Pessin J.E. Bell G.I. Annu. Rev. Physiol. 1992; 54: 911-930Crossref PubMed Scopus (385) Google Scholar). The GLUT protein family belongs to the major facilitator superfamily, the largest superfamily of proteins that function as membrane transporters (4Pao S.S. Paulsen I.T. Saier Jr., M.H. Microbiol. Mol. Biol. Rev. 1998; 62: 1-34Crossref PubMed Google Scholar). GLUT1, also known as the red cell glucose transporter, is perhaps the most extensively studied of all membrane transporters (5Mueckler M. Hresko R.C. Sato M. Biochem. Soc. Trans. 1997; 25: 951-954Crossref PubMed Scopus (33) Google Scholar). Kinetic analyses of glucose transport in the human red blood cell are mostly consistent with a simple alternating conformation mechanism (6Lowe A.G. Walmsley A.R. Agre P. Parker J.C. Red Blood Cell Membranes. Vol. 11. Marcel Dekker, Inc., New York1989: 597-634Google Scholar). Although kinetic anomalies have been observed in human erythrocytes that appear to be inconsistent with this simple mechanism (7Carruthers A. Physiol. Rev. 1990; 70: 1135-1176Crossref PubMed Scopus (329) Google Scholar, 8Cloherty E.K. Heard K.S. Carruthers A. Biochemistry. 1996; 35: 10411-10421Crossref PubMed Scopus (69) Google Scholar), an alternate explanation is that the anomalies may be the result of difficulties in accurately measuring steady-state kinetic properties in the red cell. GLUT1 was predicted to possess 12 transmembrane helices based on hydrophobicity analysis of the deduced amino acid sequence (9Mueckler M. Caruso C. Baldwin S.A. Panico M. Blench I. Morris H.R. Allard W.J. Lienhard G.E. Lodish H.F. Science. 1985; 229: 941-945Crossref PubMed Scopus (1149) Google Scholar). This prediction has been confirmed by glycosylation-scanning mutagenesis (10Hresko R.C. Kruse M. Strube M. Mueckler M. J. Biol. Chem. 1994; 269: 20482-20488Abstract Full Text PDF PubMed Google Scholar). Several of the 12 proposed transmembrane segments are predicted to form amphipathic α-helices, an observation which led to the hypothesis that these helices form the walls of a water-filled chamber through which glucose permeates the lipid bilayer (9Mueckler M. Caruso C. Baldwin S.A. Panico M. Blench I. Morris H.R. Allard W.J. Lienhard G.E. Lodish H.F. Science. 1985; 229: 941-945Crossref PubMed Scopus (1149) Google Scholar). It was also proposed that the hydroxyl- and amide-containing amino acid side chains within these helices form the sugar binding site(s) of GLUT1 via hydrogen bond formation with glucose hydroxyl groups, although hydrophobic interactions between aromatic amino acid side chains and the C-6 region of glucose also appear to be important (11Barnett J.E. Holman G.D. Munday K.A. Biochem. J. 1973; 131: 211-221Crossref PubMed Scopus (179) Google Scholar). Considerable experimental support for this structural model has accumulated. Cysteine-scanning mutagenesis and substituted cysteine accessibility studies implicate transmembrane segments 2 (12Olsowski A. Monden I. Krause G. Keller K. Biochemistry. 2000; 39: 2469-2474Crossref PubMed Scopus (48) Google Scholar), 5 (13Mueckler M. Makepeace C. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), 7 (12Olsowski A. Monden I. Krause G. Keller K. Biochemistry. 2000; 39: 2469-2474Crossref PubMed Scopus (48) Google Scholar, 14Hruz P.W. Mueckler M.M. J. Biol. Chem. 1999; 274: 36176-36180Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), 10 (15Mueckler M. Makepeace C. J. Biol. Chem. 2002; 277: 3498-3503Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), and 11 (16Hruz P.W. Mueckler M.M. Biochemistry. 2000; 39: 9367-9372Crossref PubMed Scopus (34) Google Scholar) of GLUT1 in the formation of a water-accessible cleft within the membrane. Glutamine 161 within helix 5 (17Mueckler M. Weng W. Kruse M. J. Biol. Chem. 1994; 269: 20533-20538Abstract Full Text PDF PubMed Google Scholar) and glutamine 282 within helix 7 (18Hashiramoto M. Kadowaki T. Clark A.E. Muraoka A. Momomura K. Sakura H. Tobe K. Akanuma Y. Yazaki Y. Holman G.D. J. Biol. Chem. 1992; 267: 17502-17507Abstract Full Text PDF PubMed Google Scholar) appear to participate in forming the exofacial substrate binding site. Valine 165, which is positioned one helical turn distant from glutamine 161, is accessible to aqueous sulfhydryl reagents and appears to be near the exofacial substrate binding site based on mutagenesis and inhibitor studies (19Mueckler M. Makepeace C. J. Biol. Chem. 1997; 272: 30141-30146Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). An aromatic side chain at position 412 within helix 11 appears to be essential for transport activity (20Garcia J.C. Strube M. Leingang K. Keller K. Mueckler M.M. J. Biol. Chem. 1992; 267: 7770-7776Abstract Full Text PDF PubMed Google Scholar). Finally, hydrogen exchange studies demonstrate that 30% of peptide hydrogen atoms are exposed to water in purified reconstituted GLUT1, which is consistent with the presence of an aqueous cleft in the membrane (21Jung E.K. Chin J.J. Jung C.Y. J. Biol. Chem. 1986; 261: 9155-9160Abstract Full Text PDF PubMed Google Scholar). In this study cysteine-scanning mutagenesis was used in conjunction with a sulfhydryl-specific chemical reagent to examine the possible role of transmembrane segment 8 in the formation of the GLUT1 substrate translocation pathway. Our results suggest that transmembrane segment 8 is an amphipathic α-helix with a water-accessible face that lines the exofacial portion of the sugar permeation pathway. Materials—Xenopus laevis imported African frogs were purchased from Xenopus Express (Homosassa, FL). 2-[3H]Deoxyglucose and diguanosine triphosphate (mRNA cap) were purchased from Amersham Biosciences. A Megascript™ RNA synthesis kit was purchased from Ambion, Inc. (Austin, TX), and a Transformer™ site-directed mutagenesis kit was obtained from Clontech. General Procedures—Procedures for the site-directed mutagenesis and sequencing of human GLUT1 cDNA, the in vitro transcription and purification of GLUT1 mRNAs (22Hresko R.C. Murata H. Marshall B.A. Mueckler M. J. Biol. Chem. 1994; 269: 32110-32119Abstract Full Text PDF PubMed Google Scholar), isolation, microinjection, and incubation of Xenopus oocytes (23Marshall B.A. Murata H. Hresko R.C. Mueckler M. J. Biol. Chem. 1993; 268: 26193-26199Abstract Full Text PDF PubMed Google Scholar), the preparation of purified oocyte plasma membranes and indirect immunofluorescence laser confocal microscopy (20Garcia J.C. Strube M. Leingang K. Keller K. Mueckler M.M. J. Biol. Chem. 1992; 267: 7770-7776Abstract Full Text PDF PubMed Google Scholar), SDS-polyacrylamide gel electrophoresis and immunoblotting with GLUT1 C-terminal antibody (17Mueckler M. Weng W. Kruse M. J. Biol. Chem. 1994; 269: 20533-20538Abstract Full Text PDF PubMed Google Scholar), and 2-deoxyglucose uptake measurements (24Keller K. Strube M. Mueckler M. J. Biol. Chem. 1989; 264: 18884-18889Abstract Full Text PDF PubMed Google Scholar) have been described in detail previously. Treatment with pCMBS—Stage 5 Xenopus oocytes were injected with 50 ng of wild-type or mutant GLUT1 mRNA. Two days after injection, groups of ∼20 oocytes were incubated for 15 min in the presence or absence of the indicated concentrations of p-chloromercuribenzenesulfonate (pCMBS), 1The abbreviations used are: pCMBS, p-chloromercuribenzenesulfonate; C-less, a GLUT1 molecule in which all six native cysteine residues were changed to either glycine or serine; single-C, a GLUT1 mutant constructed using the C-less parent in which a single cysteine mutation was introduced in place of one the transmembrane residues. in Barth's saline at 22 °C. The 100× concentrated reagent stock was prepared in 100% dimethyl sulfoxide, and control oocytes were treated with the appropriate concentration of vehicle alone. After a 15-min incubation period, the oocytes were washed four times in Barth's saline and then used for the determination of 2-[3H]deoxyglucose uptake (50 μm, 30 min at 22 °C). Specific Activity Determinations—Plasma membranes were prepared 3 days following the injection of 50 ng of mutant RNA/oocyte. Western blot analysis of each of the mutant transporters was performed on ∼1 μg of total membrane protein, and the intensity of the glycosylated GLUT1 band was quantified by scanning densitometry using a Molecular Dynamics phosphorimager SI. Analysis was performed using the ImageQuant NT program (Version 4.0). 2-[3H]Deoxyglucose uptake (pmol/oocyte/30 min) of each mutant was concomitantly determined in each set of experiments. Specific activity is expressed as the 2-deoxyglucose uptake/ng of mutant GLUT1 protein expressed/μg of total oocyte membrane protein. Purified human erythrocyte membranes were loaded on the same gels as the oocyte membrane samples for use as a quantitative standard. Statistical Analysis—Uptake data were analyzed for statistical significance using the two-tailed unpaired Student's t test. We described previously (19Mueckler M. Makepeace C. J. Biol. Chem. 1997; 272: 30141-30146Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) the properties of a cysteine-less (C-less) human GLUT1 polypeptide in which all six native cysteine residues were changed to either serine or glycine residues. When expressed in Xenopus oocytes the C-less transporter exhibits transport activity nearly indistinguishable from wild-type GLUT1 (19Mueckler M. Makepeace C. J. Biol. Chem. 1997; 272: 30141-30146Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 25Wellner M. Monden I. Keller K. FEBS Lett. 1995; 370: 19-22Crossref PubMed Scopus (22) Google Scholar), indicating that none of the native cysteine residues plays an essential role in transport function. C-less GLUT1 cDNA was used as a template to construct single-C mutants for transmembrane segment 8. Mutant cDNAs were constructed using oligonucleotide-mediated site-directed mutagenesis in which each of the 21 residues within transmembrane segment 8 was individually changed to a cysteine residue producing 21 mutant GLUT1 molecules, each possessing only a single cysteine residue (see Table I).Table ICysteine scanning mutagenesis of helix 8 cDNA encoding cysteine-less human Glut1 was subjected to oligonucleotide-mediated, site-directed mutagenesis, creating a series of 21 mutant cDNAs in which each of the 21 residues within transmembrane helix 8 was individually changed to cysteine. Residue number refers to the amino acid numbering for human Glut1 given in Ref. 9Mueckler M. Caruso C. Baldwin S.A. Panico M. Blench I. Morris H.R. Allard W.J. Lienhard G.E. Lodish H.F. Science. 1985; 229: 941-945Crossref PubMed Scopus (1149) Google Scholar. Amino acids are designated by the single-letter code.Residue No.Amino acid changeCodon change307Val → CysGTG → TGC308Tyr → CysTAT → TGT309Ala → CysGCC → TGC310Thr → CysACC → TGC311Ile → CysATT → TGT312Gly → CysGGC → TGC313Ser → CysTCC → TGC314Gly → CysGGT → TGT315Ile → CysATC → TGC316Val → CysGTC → TGC317Asn → CysAAC → TGC318Thr → CysACG → TGC319Ala → CysGCC → TGC320Phe → CysTTC → TGC321Thr → CysACT → TGC322Val → CysGTC → TGC323Val → CysGTG → TGC324Ser → CysTCG → TGC325Leu → CysCTG → TGC326Phe → CysTTT → TGT327Val → CysGTG → TGC Open table in a new tab Expression of the single-C mutants in the oocyte plasma membrane was confirmed by indirect immunofluorescence laser confocal microscopy (Fig. 1a) and quantitated by Western blot analysis of purified oocyte membranes (Fig. 1b). As we have observed for the analysis of other GLUT1 helices (13Mueckler M. Makepeace C. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 14Hruz P.W. Mueckler M.M. J. Biol. Chem. 1999; 274: 36176-36180Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 15Mueckler M. Makepeace C. J. Biol. Chem. 2002; 277: 3498-3503Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 16Hruz P.W. Mueckler M.M. Biochemistry. 2000; 39: 9367-9372Crossref PubMed Scopus (34) Google Scholar), the single-C mutants were expressed at varying concentrations in the oocyte plasma membrane necessitating the normalization of uptake data to expression levels to directly compare the catalytic activities of the mutants with the C-less parent. The expression of 20 of the 21 single-C mutants was detected readily in the oocytes, but the V316C mutant was expressed at levels too low to enable further analysis. Transport activity above the very low endogenous oocyte background level was detectable for all 20 mutants as determined by uptake of 2-[3H]deoxyglucose. The absolute uptake data are shown in Fig. 2a, and the specific transport activities normalized to the plasma membrane content of each mutant are presented in Fig. 2b. Cysteine substitution at threonine 310, asparagine 317, and threonine 318 significantly reduced specific transport activity relative to the C-less parent, whereas cysteine substitution at isoleucine 311 increased specific transport activity. To determine which transmembrane residues are accessible to the external aqueous solvent and may therefore comprise part of the sugar permeation pathway, transport activity was measured for each of the 21 mutants after incubation in the presence of the membrane-impermeant sulfhydryl-specific reagent, pCMBS (Fig. 3). We have demonstrated previously (19Mueckler M. Makepeace C. J. Biol. Chem. 1997; 272: 30141-30146Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) that pCMBS can permeate the glucose permeation pathway of GLUT1 and has close access to the exofacial sugar binding site. Fig. 3 presents the transport activities observed in the presence of pCMBS normalized for each mutant to the activity measured in the absence of the reagent, i.e. a value of 1 indicates no effect of pCMBS, values greater than 1 indicate stimulation by pCMBS, and values less than 1 indicate inhibition by pCMBS. The activity of four single-C mutants (A309C, T310C, S313C, and G314C) was significantly inhibited after incubation with pCMBS, indicating that the corresponding amino acid side chains reacted with the pCMBS and therefore must be accessible to the external aqueous solvent. V165C represents a well characterized positive control for inhibition by pCMBS (13Mueckler M. Makepeace C. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 19Mueckler M. Makepeace C. J. Biol. Chem. 1997; 272: 30141-30146Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Helical wheel analysis of the results of the pCMBS inhibition experiments revealed that the four residues accessible to pCMBS from the external aqueous solvent are clustered together along one face of a putative α-helix formed by transmembrane segment 8 (see Fig. 4). These results are similar to those obtained with helices 2 (12Olsowski A. Monden I. Krause G. Keller K. Biochemistry. 2000; 39: 2469-2474Crossref PubMed Scopus (48) Google Scholar), 5 (13Mueckler M. Makepeace C. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), 10 (15Mueckler M. Makepeace C. J. Biol. Chem. 2002; 277: 3498-3503Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), and 11 (16Hruz P.W. Mueckler M.M. Biochemistry. 2000; 39: 9367-9372Crossref PubMed Scopus (34) Google Scholar). Helix 7 appears to be unique in that it contains residues sensitive to pCMBS along its entire circumference, implying that its N-terminal half is completely immersed in solvent (14Hruz P.W. Mueckler M.M. J. Biol. Chem. 1999; 274: 36176-36180Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Not unexpectedly, the four residues within helix 8 that are clearly reactive to pCMBS all lie within the exoplasmic half of the helix, a result similar to that observed with helices 1, 2, 5, 7, 10, and 11, all of which possess residues close to the exoplasmic face that are accessible to the pCMBS present in the external solvent. Cysteine substitution at three positions (threonine 310, asparagine 317, and threonine 318) caused significant reductions in the intrinsic transport activity of GLUT1. The side chains of threonine 310 and asparagine 317 are predicted to lie within the aqueous translocation pathway based on the presumed orientation of the helix according to pCMBS sensitivity, suggesting that these residues may be directly involved in hydrogen bonding to glucose lying in the exofacial binding pocket. However, pCMBS reactivity only inhibited transport at position 310, which is inconsistent with hydrogen bonding between glucose and the side chain of asparagine 317. Because asparagine 317 lies further toward the cytoplasmic face of the membrane than any of the amino acid positions that were sensitive to pCMBS reactivity, it is possible that helix 8 twists as it passes through the membrane such that asparagine 317 actually abuts an adjacent helix (see Fig. 5a). The serine side chain of this residue may be involved in stabilizing helical packing via hydrogen bonding to a residue in an adjacent helix (5Mueckler M. Hresko R.C. Sato M. Biochem. Soc. Trans. 1997; 25: 951-954Crossref PubMed Scopus (33) Google Scholar or 10Hresko R.C. Kruse M. Strube M. Mueckler M. J. Biol. Chem. 1994; 269: 20482-20488Abstract Full Text PDF PubMed Google Scholar). Likewise, a cysteine substitution at threonine 318 may disrupt hydrogen bonding between the hydroxyl group of the threonine side chain and a side chain from an abutting helix. Interestingly, a cysteine substitution at isoleucine 311 significantly increased specific transport activity. This residue is predicted to either be in contact with the lipid or to abut helix 5 (see Fig. 5b). It is possible that the bulky isoleucine side chain at this position slightly impairs the movement of helices 5 and 8 associated with the transporter cycle and that this is reflected by increased activity observed upon substitution with the much more compact cysteine side chain. A major breakthrough in our understanding of the structure of the major facilitator transporters was reported recently in the form of x-ray crystallographic data at near-atomic resolution for the Escherichia coli lac permease (26Abramson J. Smirnova I. Kasho V. Verner G. Kaback H.R. Iwata S. Science. 2003; 301: 610-615Crossref PubMed Scopus (1229) Google Scholar) and the glycerol-3-P antiporter (27Huang Y. Lemieux M.J. Song J. Auer M. Wang D.N. Science. 2003; 301: 616-620Crossref PubMed Scopus (857) Google Scholar). Both molecules were crystallized in their cytoplasmic facing orientations, and in this form, the molecules exhibit a very similar basic folding pattern with a pseudo-2-fold axis of symmetry. The data for these two members of the major facilitator superfamily are consistent with the 12-transmembrane helical model that was first proposed for GLUT1 (9Mueckler M. Caruso C. Baldwin S.A. Panico M. Blench I. Morris H.R. Allard W.J. Lienhard G.E. Lodish H.F. Science. 1985; 229: 941-945Crossref PubMed Scopus (1149) Google Scholar). The pseudosymmetry also supports the notion that the 12 transmembrane transporter genes are descended from a common 6 transmembrane-encoding ancestral gene that underwent a duplication event. Based on the crystallographic data, 8 of the 12 transmembrane helices form a central cavity containing the cytoplasmic substrate binding site. Remarkably, the structures suggest analogous mechanisms for cotransport and antiport and, by inference, simple uniport involving the tilting of helices such that substrate binding sites are alternately exposed to either the cytoplasm or exoplasm. An alternating conformation mechanism of this type was originally postulated by Vidaver (28Vidaver G.A. J. Theor. Biol. 1966; 10: 301-306Crossref PubMed Scopus (84) Google Scholar) nearly four decades ago based purely on kinetic considerations, and inhibitor (29Barnett J.E. Holman G.D. Chalkley R.A. Munday K.A. Biochem. J. 1975; 145: 417-429Crossref PubMed Scopus (128) Google Scholar) and spectroscopic studies (30Appleman J.R. Lienhard G.E. Biochemistry. 1989; 28: 8221-8227Crossref PubMed Scopus (59) Google Scholar, 31Appleman J.R. Lienhard G.E. J. Biol. Chem. 1985; 260: 4575-4578Abstract Full Text PDF PubMed Google Scholar) on the red cell glucose transporter have strongly supported such a mechanism. All of the cysteine accessibility experiments (12Olsowski A. Monden I. Krause G. Keller K. Biochemistry. 2000; 39: 2469-2474Crossref PubMed Scopus (48) Google Scholar, 13Mueckler M. Makepeace C. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 14Hruz P.W. Mueckler M.M. J. Biol. Chem. 1999; 274: 36176-36180Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 15Mueckler M. Makepeace C. J. Biol. Chem. 2002; 277: 3498-3503Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 16Hruz P.W. Mueckler M.M. Biochemistry. 2000; 39: 9367-9372Crossref PubMed Scopus (34) Google Scholar) on GLUT1 employing pCMBS to detect solvent-accessible helical faces pertain to the exofacial conformation of the transporter, and thus the available crystallographic data for the lac permease and the glycerol-3-P antiporter may not be directly applicable to these GLUT1 data. However, it is likely that GLUT1 shares the same basic helical packing arrangement with these two bacterial transporters and that this arrangement is maintained during the transport cycle, i.e. major shifts in the relative arrangement of helices in the plane of the membrane do not occur. Given this assumption, we can use homology modeling to update our crude two-dimensional model for the exofacial substrate binding site of GLUT1. Helices 2 (12Olsowski A. Monden I. Krause G. Keller K. Biochemistry. 2000; 39: 2469-2474Crossref PubMed Scopus (48) Google Scholar), 5 (13Mueckler M. Makepeace C. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), 8 (this work), 10 (15Mueckler M. Makepeace C. J. Biol. Chem. 2002; 277: 3498-3503Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), and 11 (16Hruz P.W. Mueckler M.M. Biochemistry. 2000; 39: 9367-9372Crossref PubMed Scopus (34) Google Scholar) of GLUT1 all have a single discrete solvent-accessible face as defined by substituted cysteine accessibility analysis, whereas helices 1 2K. Keller, personal communication. and 7 (14Hruz P.W. Mueckler M.M. J. Biol. Chem. 1999; 274: 36176-36180Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) have solvent-accessible residues along their entire cross-sectional perimeter (14Hruz P.W. Mueckler M.M. J. Biol. Chem. 1999; 274: 36176-36180Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). These experimental observations are completely consistent with the helical packing of the lac permease (26Abramson J. Smirnova I. Kasho V. Verner G. Kaback H.R. Iwata S. Science. 2003; 301: 610-615Crossref PubMed Scopus (1229) Google Scholar) and glycerol-3-P antiporter (27Huang Y. Lemieux M.J. Song J. Auer M. Wang D.N. Science. 2003; 301: 616-620Crossref PubMed Scopus (857) Google Scholar) in their cytoplasmic facing orientations, suggesting that the transmembrane helices do not rotate substantially in the plane of the membrane during conversion to the outward facing form. The central solvent-accessible cavity is proposed to be formed by helices 1, 2, 4, and 5 in the N-terminal half of the molecule and by the analogous helices 7, 8, 10, and 11 in the C-terminal half. A ribbon diagram of the transmembrane helices as shown from the cytoplasmic face of the membrane and in the cytoplasmic facing orientation, based on homology modeling using the lac permease structure as a template, is shown in Fig. 5a. A putative model of the exofacial binding pocket is shown in Fig. 5b. Residues within helices 5, 7, and 11 have been implicated in exofacial substrate binding (reviewed in Ref. 32Hruz P.W. Mueckler M.M. Mol. Membr. Biol. 2001; 18: 183-193Crossref PubMed Scopus (139) Google Scholar). Glucose is known to be stabilized in the exofacial binding pocket via interactions between GLUT1 and the hydroxyls at positions 1, 3, and 4 (11Barnett J.E. Holman G.D. Munday K.A. Biochem. J. 1973; 131: 211-221Crossref PubMed Scopus (179) Google Scholar). The orientation of helices 5 and 7 in the model is consistent with hydrogen bond formation between hydroxyl groups of a dehydrated glucose molecule lying in the exofacial binding pocket and two residues directly implicated in substrate binding, glutamine 161 (17Mueckler M. Weng W. Kruse M. J. Biol. Chem. 1994; 269: 20533-20538Abstract Full Text PDF PubMed Google Scholar) and glutamine 282 (18Hashiramoto M. Kadowaki T. Clark A.E. Muraoka A. Momomura K. Sakura H. Tobe K. Akanuma Y. Yazaki Y. Holman G.D. J. Biol. Chem. 1992; 267: 17502-17507Abstract Full Text PDF PubMed Google Scholar). Glutamine 282 appears to interact directly with the glucose C-1 hydroxyl group (33Seatter M.J. De la Rue S.A. Porter L.M. Gould G.W. Biochemistry. 1998; 37: 1322-1326Crossref PubMed Scopus (94) Google Scholar). The orientation of helix 5 is also consistent with the observation that valine 165 lies near the outer vestibule of the exofacial substrate binding site, although this residue is not directly involved in transport activity (19Mueckler M. Makepeace C. J. Biol. Chem. 1997; 272: 30141-30146Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Site-directed mutagenesis studies (19Mueckler M. Makepeace C. J. Biol. Chem. 1997; 272: 30141-30146Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) have demonstrated that side chains bulkier than valine are not tolerated at position 165, whereas smaller side chains are tolerated. The orientation of valine 165 in the model is consistent with bulky substitutions at this position interfering with hydrogen bond formation between glucose and glutamine 161. The low transport activity of the T310C and N317C mutants along with their orientation along the solvent-exposed face of helix 8 suggests that the side chains of these residues may be involved in hydrogen bond formation with glucose in the exofacial substrate binding site. The orientation of helix 11 is consistent with a hydrophobic interaction between the C-6 region of glucose and tryptophan 412. A hydrophobic interaction, possibly a stacking interaction involving an aromatic ring of GLUT1 and the C-6 region of glucose, was predicted by Barnett et al. (11Barnett J.E. Holman G.D. Munday K.A. Biochem. J. 1973; 131: 211-221Crossref PubMed Scopus (179) Google Scholar) based on transport studies employing substituted glucose analogs. Site-directed mutagenesis studies have shown that a tryptophan at position 412 is critical for transport activity (20Garcia J.C. Strube M. Leingang K. Keller K. Mueckler M.M. J. Biol. Chem. 1992; 267: 7770-7776Abstract Full Text PDF PubMed Google Scholar), and additional studies indicate that an aromatic ring is essential at this position. 3M. Mueckler, unpublished observations. Determination of the structure of GLUT1 in the outward facing configuration awaits the crystallization of the protein, a feat that has not yet been achieved for any eukaryotic membrane protein. Although the recent successes with the bacterial transporters are encouraging, the problems faced with eukaryotic membrane proteins, including structural heterogeneity because of post-translational modifications and the lack of robust overexpression systems, are extraordinarily daunting. In the interim, various biochemical approaches can be used to test the applicability of homology modeling based on the structures of the lac permease and glycerol-3-P antiporter.
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