Analysis of Glucose Transporter Topology and Structural Dynamics
2008; Elsevier BV; Volume: 283; Issue: 52 Linguagem: Inglês
10.1074/jbc.m804802200
ISSN1083-351X
AutoresDavid M. Blodgett, Christopher A. Graybill, Anthony Carruthers,
Tópico(s)Plant nutrient uptake and metabolism
ResumoHomology modeling and scanning cysteine mutagenesis studies suggest that the human glucose transport protein GLUT1 and its distant bacterial homologs LacY and GlpT share similar structures. We tested this hypothesis by mapping the accessibility of purified, reconstituted human erythrocyte GLUT1 to aqueous probes. GLUT1 contains 35 potential tryptic cleavage sites. Fourteen of 16 lysine residues and 18 of 19 arginine residues were accessible to trypsin. GLUT1 lysine residues were modified by isothiocyanates and N-hydroxysuccinimide (NHS) esters in a substrate-dependent manner. Twelve lysine residues were accessible to sulfo-NHS-LC-biotin. GLUT1 trypsinization released full-length transmembrane helix 1, cytoplasmic loop 6–7, and the long cytoplasmic C terminus from membranes. Trypsin-digested GLUT1 retained cytochalasin B and d-glucose binding capacity and released full-length transmembrane helix 8 upon cytochalasin B (but not d-glucose) binding. Transmembrane helix 8 release did not abrogate cytochalasin B binding. GLUT1 was extensively proteolyzed by α-chymotrypsin, which cuts putative pore-forming amphipathic α-helices 1, 2, 4, 7, 8, 10, and 11 at multiple sites to release transmembrane peptide fragments into the aqueous solvent. Putative scaffolding membrane helices 3, 6, 9, and 12 are strongly hydrophobic, resistant to α-chymotrypsin, and retained by the membrane bilayer. These observations provide experimental support for the proposed GLUT1 architecture; indicate that the proposed topology of membrane helices 5, 6, and 12 requires adjustment; and suggest that the metastable conformations of transmembrane helices 1 and 8 within the GLUT1 scaffold destabilize a sugar translocation intermediate. Homology modeling and scanning cysteine mutagenesis studies suggest that the human glucose transport protein GLUT1 and its distant bacterial homologs LacY and GlpT share similar structures. We tested this hypothesis by mapping the accessibility of purified, reconstituted human erythrocyte GLUT1 to aqueous probes. GLUT1 contains 35 potential tryptic cleavage sites. Fourteen of 16 lysine residues and 18 of 19 arginine residues were accessible to trypsin. GLUT1 lysine residues were modified by isothiocyanates and N-hydroxysuccinimide (NHS) esters in a substrate-dependent manner. Twelve lysine residues were accessible to sulfo-NHS-LC-biotin. GLUT1 trypsinization released full-length transmembrane helix 1, cytoplasmic loop 6–7, and the long cytoplasmic C terminus from membranes. Trypsin-digested GLUT1 retained cytochalasin B and d-glucose binding capacity and released full-length transmembrane helix 8 upon cytochalasin B (but not d-glucose) binding. Transmembrane helix 8 release did not abrogate cytochalasin B binding. GLUT1 was extensively proteolyzed by α-chymotrypsin, which cuts putative pore-forming amphipathic α-helices 1, 2, 4, 7, 8, 10, and 11 at multiple sites to release transmembrane peptide fragments into the aqueous solvent. Putative scaffolding membrane helices 3, 6, 9, and 12 are strongly hydrophobic, resistant to α-chymotrypsin, and retained by the membrane bilayer. These observations provide experimental support for the proposed GLUT1 architecture; indicate that the proposed topology of membrane helices 5, 6, and 12 requires adjustment; and suggest that the metastable conformations of transmembrane helices 1 and 8 within the GLUT1 scaffold destabilize a sugar translocation intermediate. The major facilitator superfamily (MFS) 2The abbreviations used are: MFSmajor facilitator superfamilyC-Abrabbit polyclonal antiserum raised against a synthetic peptide comprising GLUT1 residues 480–492CBcytochalasin BDMSOdimethylsulfoxideELISAenzyme-linked immunosorbent assayEmrDthe multidrug transporter of Escherichia coliGlpTglycerol 3-phosphate glycerol antiporter of E. coliGLUT1human erythrocyte glucose transport proteinLacYthe lactate: proton symporter of E. coliMSmass spectrometryOxlToxalate transport of Oxalobacter formigenesRhDRh blood group D antigenHPLChigh performance liquid chromatographyESIelectrospray ionizationsulfo-NHS-LC-biotinsulfosuccinimidyl-6-(biotinamido)hexanoateTMtransmembrane domainNHSN-hydroxysuccinimideLloop 2The abbreviations used are: MFSmajor facilitator superfamilyC-Abrabbit polyclonal antiserum raised against a synthetic peptide comprising GLUT1 residues 480–492CBcytochalasin BDMSOdimethylsulfoxideELISAenzyme-linked immunosorbent assayEmrDthe multidrug transporter of Escherichia coliGlpTglycerol 3-phosphate glycerol antiporter of E. coliGLUT1human erythrocyte glucose transport proteinLacYthe lactate: proton symporter of E. coliMSmass spectrometryOxlToxalate transport of Oxalobacter formigenesRhDRh blood group D antigenHPLChigh performance liquid chromatographyESIelectrospray ionizationsulfo-NHS-LC-biotinsulfosuccinimidyl-6-(biotinamido)hexanoateTMtransmembrane domainNHSN-hydroxysuccinimideLloop of transport proteins comprises more than 1,000 proteins that mediate passive and secondary active transfer of small molecules across membranes (1Saier Jr., M.H. Beatty J.T. Goffeau A. Harley K.T. Heijne W.H. Huang S.C. Jack D.L. Jahn P.S. Lew K. Liu J. Pao S.S. Paulsen I.T. Tseng T.T. Virk P.S. J. Mol. Microbiol. Biotechnol. 1999; 1: 257-279PubMed Google Scholar). The facilitative glucose transport proteins (GLUT1–12) catalyze monosaccharide uniport in vertebrates (2Joost H.G. Bell G.I. Best J.D. Birnbaum M.J. Charron M.J. Chen Y.T. Doege H. James D.E. Lodish H.F. Moley K.H. Moley J.F. Mueckler M. Rogers S. Schurmann A. Seino S. Thorens B. Am. J. Physiol. 2002; 282: E974-E976Crossref PubMed Scopus (324) Google Scholar) and display tissue-specific isoform expression. GLUT1 is expressed in most tissues but is especially abundant in the circulatory system (3Joost H.G. Thorens B. Mol. Membr. Biol. 2001; 18: 247-256Crossref PubMed Scopus (560) Google Scholar) and at blood-tissue barriers such as the blood-brain barrier (4Guo X. Geng M. Du G. Biochem. Genet. 2005; 43: 175-187Crossref PubMed Scopus (75) Google Scholar). major facilitator superfamily rabbit polyclonal antiserum raised against a synthetic peptide comprising GLUT1 residues 480–492 cytochalasin B dimethylsulfoxide enzyme-linked immunosorbent assay the multidrug transporter of Escherichia coli glycerol 3-phosphate glycerol antiporter of E. coli human erythrocyte glucose transport protein the lactate: proton symporter of E. coli mass spectrometry oxalate transport of Oxalobacter formigenes Rh blood group D antigen high performance liquid chromatography electrospray ionization sulfosuccinimidyl-6-(biotinamido)hexanoate transmembrane domain N-hydroxysuccinimide loop major facilitator superfamily rabbit polyclonal antiserum raised against a synthetic peptide comprising GLUT1 residues 480–492 cytochalasin B dimethylsulfoxide enzyme-linked immunosorbent assay the multidrug transporter of Escherichia coli glycerol 3-phosphate glycerol antiporter of E. coli human erythrocyte glucose transport protein the lactate: proton symporter of E. coli mass spectrometry oxalate transport of Oxalobacter formigenes Rh blood group D antigen high performance liquid chromatography electrospray ionization sulfosuccinimidyl-6-(biotinamido)hexanoate transmembrane domain N-hydroxysuccinimide loop GLUT1 comprises 492 amino acids; is hydrophobic; contains a single, exofacial N-linked glycosylation site (5Mueckler 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 (1126) Google Scholar); and is predominantly α-helical (6Cairns 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). Hydropathy analysis (5Mueckler 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 (1126) Google Scholar), scanning glycosylation mutagenesis (7Hresko R.C. Kruse M. Strube M. Mueckler M. J. Biol. Chem. 1994; 269: 20482-20488Abstract Full Text PDF PubMed Google Scholar), proteolysis, antibody binding, and covalent modification studies indicate that GLUT1 contains intracellular N and C termini and 12 transmembrane domain (TM) α-helices (8Hruz P.W. Mueckler M.M. Mol. Membr. Biol. 2001; 18: 183-193Crossref PubMed Scopus (136) Google Scholar). Amphipathic α-helices are proposed to form an aqueous translocation pathway for glucose transport across the plasma membrane (9Jung E.K. Chin J.J. Jung C.Y. J. Biol. Chem. 1986; 261: 9155-9160Abstract Full Text PDF PubMed Google Scholar, 10Mueckler M. Makepeace C. J. Biol. Chem. 2006; 281: 36993-36998Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 11Salas-Burgos A. Iserovich P. Zuniga F. Vera J.C. Fischbarg J. Biophys. J. 2004; 87: 2990-2999Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). However, the detailed three-dimensional structure of GLUT1 is not known, and GLUT1 conformational changes catalyzing transport are unclear. The structures of bacterial MFS transport proteins offer new insights into carrier structure (12Dahl S.G. Sylte I. Ravna A.W. J. Pharmacol. Exp. Ther. 2004; 309: 853-860Crossref PubMed Scopus (62) Google Scholar). The lactose permease (LacY (13Abramson J. Smirnova I. Kasho V. Verner G. Kaback H.R. Iwata S. Science. 2003; 301: 610-615Crossref PubMed Scopus (1211) Google Scholar)), the glycerol 3-phosphate antiporter (GlpT (14Huang Y. Lemieux M.J. Song J. Auer M. Wang D.N. Science. 2003; 301: 616-620Crossref PubMed Scopus (843) Google Scholar)), a multidrug transporter (EmrD (15Yin Y. He X. Szewczyk P. Nguyen T. Chang G. Science. 2006; 312: 741-744Crossref PubMed Scopus (312) Google Scholar)), and the oxalate transporter (OxlT (16Hirai T. Heymann J.A. Shi D. Sarker R. Maloney P.C. Subramaniam S. Nat. Struct. Biol. 2002; 9: 597-600PubMed Google Scholar)) display little sequence similarity but share similar structures suggesting a common MFS protein architecture. Although mammalian MFS proteins, such as GLUT1, can be refractory to three-dimensional crystallization (12Dahl S.G. Sylte I. Ravna A.W. J. Pharmacol. Exp. Ther. 2004; 309: 853-860Crossref PubMed Scopus (62) Google Scholar, 17Kasho V.N. Smirnova I.N. Kaback H.R. J. Mol. Biol. 2006; 358: 1060-1070Crossref PubMed Scopus (37) Google Scholar), they may be homology-modeled using crystallized homologs as templates (12Dahl S.G. Sylte I. Ravna A.W. J. Pharmacol. Exp. Ther. 2004; 309: 853-860Crossref PubMed Scopus (62) Google Scholar). Salas-Burgos et al. (11Salas-Burgos A. Iserovich P. Zuniga F. Vera J.C. Fischbarg J. Biophys. J. 2004; 87: 2990-2999Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) and Holyoake et al. (18Holyoake J. Caulfeild V. Baldwin S.A. Sansom M.S. Biophys. J. 2006; 91: L84-L86Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) have modeled a GLUT1 structure using the GlpT template and biochemical and mutagenesis data to validate their results. GLUT1 cysteine-scanning mutagenesis studies (10Mueckler M. Makepeace C. J. Biol. Chem. 2006; 281: 36993-36998Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) broadly support the resulting GLUT1 MFS α-helical packing arrangement but also note significant accessibility in TM regions that, according to the model, should be inaccessible (10Mueckler M. Makepeace C. J. Biol. Chem. 2006; 281: 36993-36998Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). 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 (11Salas-Burgos A. Iserovich P. Zuniga F. Vera J.C. Fischbarg J. Biophys. J. 2004; 87: 2990-2999Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), whereas biochemical studies suggest that cytochalasin B binds close to cytoplasmic loop 10–11 (19Holman G.D. Rees W.D. Biochim. Biophys. Acta. 1987; 897: 395-405Crossref PubMed Scopus (64) Google Scholar, 20Inukai K. Asano T. Katagiri H. Anai M. Funaki M. Ishihara H. Tsukuda K. Kikuchi M. Yazaki Y. Oka Y. Biochem. J. 1994; 302: 355-361Crossref PubMed Scopus (34) Google Scholar, 21Garcia J.C. Strube M. Leingang K. Keller K. Mueckler M.M. J. Biol. Chem. 1992; 267: 7770-7776Abstract Full Text PDF PubMed Google Scholar). This discrepancy is consistent with a recent demonstration that homology modeling approximates carrier topology and architecture but is less successful at defining the spatial arrangement of specific residues (22Lemieux M.J. Mol. Membr. Biol. 2007; 24: 333-341Crossref PubMed Scopus (40) Google Scholar). Deviations between experimental and modeled structures may also reflect structural and functional differences between template and target proteins. The proposed GLUT1 architecture is inherently accessible to experimental evaluation. GLUT1 contains 16 lysine and 20 arginine residues (5Mueckler 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 (1126) Google Scholar) located within proposed intra- and extracellular loops, within the N and C termini, and at the membrane/solvent interface. Water-exposed lysine residues should be accessible to primary amine-reactive, polar covalent probes. Water-exposed lysine and arginine residues may also be accessible to trypsin. This study examined the accessibility of membrane-resident GLUT1 to proteases and to primary amine-reactive covalent probes. To maximize in vivo relevance, we specifically studied human erythrocyte GLUT1 purified under conditions where native function and quaternary structure are preserved (23Graybill C. van Hoek A.N. Desai D. Carruthers A.M. Carruthers A. Biochemistry. 2006; 45: 8096-8107Crossref PubMed Scopus (21) Google Scholar). Our findings provide direct experimental support for the proposed GLUT1 architecture, suggest that the topology of some membrane helices requires minor adjustment, and illustrate that transmembrane helix 8 undergoes significant conformational change upon GLUT1 ligand binding. Materials—Fresh, de-identified human blood was from Biological Specialties Corp. (Colmar, PA). Protein assays, Pro Blue Coomassie stain, sulfo-NHS-LC-biotin, fluorescein isothiocyanate, and SuperSignal chemiluminescence kits were from Pierce. Glycerol-free endoglycosidase peptide-N-glycosidase F was from New England Biolabs (Ipswich, MA). Nitrocellulose and Immobilon-P were from Fisher Scientific. Purified rabbit IgGs raised against a synthetic cytoplasmic C-terminal peptide of human GLUT1 (C-Ab, residues 480–492) were from Animal Pharm Services, Inc. (Healdsburg, CA). All other reagents were from Sigma-Aldrich. Solutions—Saline comprised 150 mm NaCl, 10 mm Tris-HCl, and 0.5 mm EDTA, pH 7.4. Lysis medium contained 10 mm Tris-HCl and 0.2 mm EDTA, pH 7.2. Tris medium contained 50 mm Tris-HCl, pH 7.4. Kaline consisted of 150 mm KCl, 5 mm HEPES, 4 mm EGTA, and 5 mm MgCl2. Phosphate-buffered saline containing Tween comprised 140 mm NaCl, 10 mm Na2HPO4, 3.4 mm KCl, 1.84 mm KH2PO4, and 0.1% Tween, pH 7.3. Human Erythrocyte Membranes and Glucose Transport Protein—Glucose transporter and endogenous lipids were purified from human erythrocyte membranes in the absence of reductant as described previously (24Hebert D.N. Carruthers A. J. Biol. Chem. 1992; 267: 23829-23838Abstract Full Text PDF PubMed Google Scholar, 25Steck T.L. Yu J. J. Supramol. Struct. 1973; 1: 220-232Crossref PubMed Scopus (397) Google Scholar). The resulting unsealed GLUT1 proteoliposomes (26Sultzman L.A. Carruthers A. Biochemistry. 1999; 38: 6640-6650Crossref PubMed Scopus (27) Google Scholar) contain 90% GLUT1, 8% RhD protein, and 2% nucleoside transporter (by protein mass) and contain equal masses of lipid and protein (27Zottola R.J. Cloherty E.K. Coderre P.E. Hansen A. Hebert D.N. Carruthers A. Biochemistry. 1995; 34: 9734-9747Crossref PubMed Scopus (114) Google Scholar). Cytochalasin B Binding—[3H]cytochalasin B (CB) binding to GLUT1 proteoliposomes was measured as described previously (23Graybill C. van Hoek A.N. Desai D. Carruthers A.M. Carruthers A. Biochemistry. 2006; 45: 8096-8107Crossref PubMed Scopus (21) Google Scholar). GLUT1 Deglycosylation—GLUT1 (50 μgin50 μl of kaline buffer) was incubated with 1,500 activity units of peptide-N-glycosidase F for 1 h at 37 °C. Gel Electrophoresis and Western Blotting—GLUT1 protein was resolved on 8% polyacrylamide gels (28Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205523) Google Scholar). Immunoblot analysis used C-Ab at 1:10,000 dilution as described previously (27Zottola R.J. Cloherty E.K. Coderre P.E. Hansen A. Hebert D.N. Carruthers A. Biochemistry. 1995; 34: 9734-9747Crossref PubMed Scopus (114) Google Scholar). GLUT1 Covalent Modification—GLUT1 (25–100 μg) was incubated with EZ-Link sulfo-NHS-LC-biotin (10 mm; pH 7.4, 4 °C), with fluorescein isothiocyanate (500 μm; pH 8, 4 °C), or with [14C]phenylisothiocyanate (50 μm; pH 8.0, 4 °C) at GLUT1:covalent probe molar ratios ranging from 1:0.2 to 1:2,000 for 1 min to 1,800 min (29Blodgett D.M. De Zutter J.K. Levine K.B. Karim P. Carruthers A. J. Gen. Physiol. 2007; 130: 157-168Crossref PubMed Scopus (52) Google Scholar). Reactions were quenched with 50 mm Tris-HCl or glycine, and membranes were sedimented, washed several times with additional quench solution, and resuspended in medium appropriate for follow-up analysis. ELISA—Biotin incorporation was quantitated by ELISA. Wells were precoated with C-Ab (200 μl of a 1:5,000 dilution; 300 min at 37 °C) and then blocked with 3% bovine serum albumin (120 min at 37 °C). Biotinylated GLUT1 was solubilized (2% Triton X-100 in phosphate-buffered saline for 60 min at 4 °C), clarified by centrifugation (≈200,000 × g for 15 min), and applied to triplicate wells (200 μl/well, 2-h incubation at 4 °C). Wells were washed and blocked in phosphate-buffered saline containing 3% bovine serum albumin and 2% Triton X-100. Horseradish peroxidase-conjugated streptavidin was added (1 μg/ml; 1 h at 20 °C), and following washing, product development was measured as absorbance at 415 nm using a Benchmark Microplate Reader (Bio-Rad). Tryptic and α-Chymotryptic Digested GLUT1 Peptides—Unmodified or pretreated reconstituted GLUT1 in kaline (0.5 mg/ml, 55-μl total reaction volume) was digested with trypsin or α-chymotrypsin at a 1:10 ratio (enzyme:GLUT1 by mass) for 0–2 h at 30 °C. Peptides were subjected to HPLC-ESI-MS/MS immediately or were processed further. Digests were separated into aqueous and membrane fractions by centrifugation for 30 min at 4 °C in a Beckman air-driven ultracentrifuge (≈200,000 × g). HPLC Separation of GLUT1 Peptides—Peptides (25 μg of GLUT1 in 50 μl of kaline) were resolved by reverse phase chromatography (30Weinglass A. Whitelegge J.P. Faull K.F. Kaback H.R. J. Biol. Chem. 2004; 279: 41858-41865Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) using a polystyrene divinylbenzene copolymer column (5 μm, 300 Å, 150 × 2.1 mm; PLRP-S, Polymer Laboratories, Amherst, MA)) at 40 °C. Peptides were eluted using one of two gradients comprising solvent A (5% 1:1 (v/v) acetonitrile:isopropanol in water) and solvent B (1:1 (v/v) acetonitrile:isopropanol). Both solvents contained 0.1% formic acid and 0.01% trifluoroacetic acid (30Weinglass A. Whitelegge J.P. Faull K.F. Kaback H.R. J. Biol. Chem. 2004; 279: 41858-41865Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 31Abe Y. Chaen T. Jin X.R. Hamasaki T. Hamasaki N. J. Biochem. (Tokyo). 2004; 136: 97-106Crossref PubMed Scopus (14) Google Scholar). Supernatant fractions were resolved using 15-min equilibration in 98% solvent A and 2% solvent B followed by raising the percentage of solvent B to 10% over 3 min, to 45% over 37 min, and to 95% over 2 min where it was held for 10 min prior to re-equilibration. The flow rate was 200 μl/min. Resolution of membrane fraction peptides required longer exposure to organic solvents: after 15 min of exposure to 95% solvent A and 5% solvent B, the percentage of solvent B was raised to 20% over 4 min and then to 95% over 56 min where it was held for 10 min prior to re-equilibration. Electrospray Ionization Mass Spectrometry—ESI-MS analysis was performed using a Thermo Fisher LCQ or an LTQ electrospray ionization mass spectrometer. Operational parameters included positive ion mode; spray voltage, 4.5 kV; capillary temperature, 225 °C; and scan range, m/z 400–2,000. MS/MS fragments of isolated peptide species were produced in the ion trap by collision-induced dissociation with helium gas at 35 V. Acquisition methods were created using Xcalibur (version 1.2). MS/MS spectra were identified using the Bioworks Sequest version 3.2 data base search program and the human red blood cell proteome data base compiled by Kakhniashvili et al. (32Kakhniashvili D.G. Bulla L.A. J. Goodman S.R. Mol. Cell. Proteomics. 2004; 3: 501-509Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Experimental System—All experiments used GLUT1 proteoliposomes comprising purified GLUT1 and co-purified, native erythrocyte lipids. These proteoliposomes are unsealed and simultaneously expose endo- and exofacial GLUT1 domains to exogenous ligands, proteolytic enzymes, and antibodies (26Sultzman L.A. Carruthers A. Biochemistry. 1999; 38: 6640-6650Crossref PubMed Scopus (27) Google Scholar, 33Appleman J.R. Lienhard G.E. Biochemistry. 1989; 28: 8221-8227Crossref PubMed Scopus (58) Google Scholar). All analyses were performed using untreated GLUT1 or GLUT1 exposed to trypsin or α-chymotrypsin for 2 h at 30 °C by which time proteolysis is complete as judged by SDS-PAGE or immunoblot analysis (29Blodgett D.M. De Zutter J.K. Levine K.B. Karim P. Carruthers A. J. Gen. Physiol. 2007; 130: 157-168Crossref PubMed Scopus (52) Google Scholar). GLUT1 residues comprising individual TM α-helices, intra- and extracellular loops, and the N and C termini were assigned according to the homology model of Salas-Burgos et al. (11Salas-Burgos A. Iserovich P. Zuniga F. Vera J.C. Fischbarg J. Biophys. J. 2004; 87: 2990-2999Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) (Fig. 1A) in which TMs are organized by location and function: those forming the aqueous translocation pathway (group 1 (TMs 1, 4, 7, and 10) and group 2 (TMs 2, 5, 8, and 11)) or those comprising the lipid bilayer-embedded scaffolding region (group 3 (TMs 3, 6, 9, and 12); Fig. 1B (13Abramson J. Smirnova I. Kasho V. Verner G. Kaback H.R. Iwata S. Science. 2003; 301: 610-615Crossref PubMed Scopus (1211) Google Scholar, 14Huang Y. Lemieux M.J. Song J. Auer M. Wang D.N. Science. 2003; 301: 616-620Crossref PubMed Scopus (843) Google Scholar)). Loops describe the TMs they connect. For example, L1–2 is the exofacial loop connecting TMs 1 and 2. GLUT1 Accessibility to Proteolytic Enzymes—GLUT1 contains 35 potential tosylphenylalanyl chloromethyl ketone-treated trypsin proteolytic sites (16 lysine and 19 arginine residues (5Mueckler 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 (1126) Google Scholar)). These residues are located within proposed intra- and extracellular loops, within the N and C termini, and at the membrane/solvent interface. Our results show that 32 sites were detected by MS/MS analysis of trypsin-treated GLUT1 (Fig. 2A). Only residues Arg153 and Lys183 flanking TM5 and Lys451 at the C-terminal end of TM12 appear to be inaccessible to trypsin digestion. α-Chymotrypsin is less specific and in our hands consistently cleaved GLUT1 at the C-terminal end of phenylalanine, tyrosine, tryptophan, leucine, alanine, methionine, and glutamic acid. These residues are located both in putative, water-exposed domains and in membrane-embedded protein domains. Each putative TM contains at least five potential α-chymotrypsin cleavage sites, but we observed TM-specific proteolysis (Fig. 2B). Although groups 1 and 2 TM α-chymotrypsin cleavage products were detected, group 3 TM proteolysis was not detected (Table 1 and Fig. 3A). The number of detected cleavage sites declined in the following order: group 1 (15 sites) > group 2 (10 sites) >> group 3 TMs (one site; see Table 1). Putative, extramembranous regions revealed more proteolytic products than membrane-embedded domains, and the longest contiguous regions of the protein (N and C termini, L1–2, and L6–7) produced twice as many cleavage products than shorter loops.TABLE 1Susceptibility of GLUT1 side chains to proteolytic attackGrouping and regionaRegions are delineated as 1) individual, membrane-embedded α-helices; 2) membrane-spanning helix groups; 3) loop, tail, and regions not embedded within the membrane; 4) summation of the individual membrane-embedded α-helices versus all other regions; and 5) the proteolytic susceptibility of the entire transport structurePotential sitesbThe potential number of cleavage sitesSites cleavedcThe number of sites detected as cleavage sitesCleavage ratedThe percent accessibility of the region to proteolysisSSReThe relative hydrophobicity of each region was calculated using the sequence specific retention (SSR) calculator developed by the Manitoba Center for Proteomics (42). For membrane-spanning α-helices, this calculation covers only peptide sequence that is embedded in the bilayer domain according to Salas-Burgos et al. (11) (Fig. 1)%1TM1745738.7TM71066055.1TM2944465.8TM8524045.8TM3131868.1TM9120065.5TM4944459.5TM10111976.3TM5722956.8TM11822546.4TM690081.4TM121200101.42Group 1 TMs (1, 4, 7, and 10)37154157.4Group 2 TMs (2, 5, 8, and 11)29103453.7Group 3 TMs (3, 6, 9, and 12)461279.13N terminus10550L1–21077044.1L6–737246554.1C terminus19105337.5Others461328Total12259484Membrane1122623Extramembrane12259485GLUT1 total2348536a Regions are delineated as 1) individual, membrane-embedded α-helices; 2) membrane-spanning helix groups; 3) loop, tail, and regions not embedded within the membrane; 4) summation of the individual membrane-embedded α-helices versus all other regions; and 5) the proteolytic susceptibility of the entire transport structureb The potential number of cleavage sitesc The number of sites detected as cleavage sitesd The percent accessibility of the region to proteolysise The relative hydrophobicity of each region was calculated using the sequence specific retention (SSR) calculator developed by the Manitoba Center for Proteomics (42Krokhin O.V. Craig R. Spicer V. Ens W. Standing K.G. Beavis R.C. Wilkins J.A. Mol. Cell. Proteomics. 2004; 3: 908-919Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). For membrane-spanning α-helices, this calculation covers only peptide sequence that is embedded in the bilayer domain according to Salas-Burgos et al. (11Salas-Burgos A. Iserovich P. Zuniga F. Vera J.C. Fischbarg J. Biophys. J. 2004; 87: 2990-2999Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) (Fig. 1) Open table in a new tab SDS-PAGE-denatured GLUT1 was more uniformly accessible to protease (Fig. 3B). Residues Arg153 and Lys183 flanking TM5 were detected as tryptic cleavage sites in SDS-PAGE gel-eluted GLUT1. CD analysis demonstrated that gel-eluted GLUT1 contains significant α-helical structure, which may explain the persistent protease resistance of strongly hydrophobic TMs in the presence of SDS. GLUT1 Peptide Water Solubility—GLUT1 proteolytic digests may be separated into membrane and aqueous fractions by centrifugation. Trypsin digestion released GLUT1 N and C termini, a portion of L1–2, and all of L6–7 into the aqueous buffer. The L1–2 peptide was detected only following GLUT1 deglycosylation by peptide-N-glycosidase F. Full-length TM1, cleaved at intracellular Arg11 and outer bilayer-hemileaflet Lys38, was also detected in the aqueous fraction (Fig. 4A). α-Chymotrypsin digestion released a similar pattern of GLUT1 extramembranous regions into the aqueous fraction including the N and C termini, a portion of L7–8, and all of L1–2, L6–7, and L10–11. The isolated aqueous fraction also contained TM1 in its entirety as well as portions of TMs 2, 4, 7, 8, 10, and 11 (Fig. 4B). TM8 (residues Ala301 through Arg330) was released into the buffer when trypsin digestion was carried out in the presence of the transport inhibitor CB (10 μm; Figs. 4A and 5) or when the membrane fraction of a CB-free trypsin digest was resuspended in buffer containing CB. Cytochalasin D, DMSO, d-glucose, and maltose were unable to displace TM8 from trypsin-digested membranes. Dot-blots of α-chymotrypsin-digested GLUT1 using peptide-directed antibodies confirmed that GLUT1 N and C termini and loop 7–8 peptides were released by chymotrypsin but that loop 8–9 peptides (residues 325–338) were released only when CB was present during proteolysis. Ligand Binding to Proteolyzed GLUT1—Trypsinization increased the CB binding capacity of GLUT1 (Table 2), increased Kd(app) for CB binding, and decreased Ki(app) for d-glucose inhibition of CB binding. Displacement of TM8 by trypsinization in the presence of 10 μm CB was without additional impact on CB or d-glucose binding. CB binding to α-chymotrypsin-treated GLUT1 was undetectable (Table 2).TABLE 2Ligand binding to GLUT1Experimenta[3H]Cytochalasin B binding to GLUT1 proteoliposomes. CB binding was measured using GLUT1 proteoliposomes in saline buffer (control); GLUT1 proteoliposomes were incubated with trypsin to remove fragments L1, L2, L6, N and C termini, and TM1 (trypsin); GLUT1 was trypsinized in the presence of 10 μm CB to additionally remove TM8 (post-trypsin/CB); and GLUT1 proteoliposomes were treated with α-chymotrypsin. Results are shown as mean ± S.E. "—" indicates that CB binding was not detectedCytochalasin B bindingbCB binding was measured in tri
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