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Structure, Function and Regulation of Glucose Transporters Membrane Group/Hormone Group Joint Colloquium Organized and Edited by G. Holman (School of Biology and Biochemistry, University of Bath) and Sponsored by Pfizer. Novo Nordisk. SmithKline Beecham and Zeneca Pharmaceuticals. 66 Ist Meeting held at Bath, 9- I I April 1997. Structure, function and biosynthesis of GLUT I M. Mueckler*, R. C. Hresko and M. Sat0 Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63 I 10, U.S.A. Structure and function of GLUT1 [9] using glycosylation-scanning mutagenesis, a procedure whereby N-linked glycosylation consensus sites are engineered into the predicted aqueous domains of a membrane protein, and the ability of a specific site to be glycosylated is interpreted as reflecting an extracytoplasmic disposition for that site. T h e absence of glycosylation at a specific site is inferred as indicating a cytoplasmic disposition, although this conclusion is necessarily more tenuous than the converse assignment. We used the glycosylated exofacial linker domain of GLUT4 as the glycosylation marker. When the glycosylation mutants were expressed in Xenopus oocytes, the pattern of glycosylation obtained with the mutants was exactly as predicted by the topological model for G L U T l that we proposed on the basis of hydropathic analysis of the deduced amino acid sequence [S]. These data thus provide strong experimental support for the original topological model. Interestingly, however, the results obtained when the mutants were expressed in reticulocyte lysate supplemented with canine pancreatic microsomes differed in a significant manner from the oocyte results. Although all of the predicted exoplasmically disposed sites were fully glycosylated in the reticulocyte system, all of the predicted cytoplasmically disposed sites showed an approx. 50% level of glycosylation. These data suggest that aberrant insertion of a fraction of the chimaeric protein molecules harbouring the glycosylation marker in cytosolic domains occurred in the in vitro system, but not in the in vivo system. Analysis of additional chimaeras demonstrated that the altered topology T h e G L U T family of 50-60 kDa membrane glycoproteins consists of five known members, four of which are involved in the facilitative transport of glucose across cellular membranes [l-31. GLUT1, the prototype member of this family, may be the most extensively studied of all mammalian membrane transporters. It was one of the first membrane transporters to be purified to homogeneity [4] and cloned [5], and its kinetic properties in the erythrocyte membrane have been studied for over four decades [6]. Despite this attention, our knowledge of the structurelfunction properties and biosynthesis of this molecule, and of membrane transporters in general, is still rudimentary. T h e cloning of human G L U T l from HepG2 cells revealed a polypeptide of 492 amino acid residues with a molecular mass of 54 117 Da [5]. In vitro translation studies indicated that G L U T l possesses a single N-linked oligosaccharide at [7]. G L U T l was the first of what is now a large superfamily [S] of membrane transport proteins that are predicted to possess 12 a-helical transmembrane segments based on hydrophobicity analysis of its deduced amino acid sequence. Until recently, however, this ubiquitous 12-transmembrane-segment topological motif was without direct experimental support and thus remained purely hypothetical. We tested the topological model for G L U T l Abbreviations used: ER, endoplasmic reticulum membrane. *To whom correspondence should be addressed. 95 I I997 Biochemical Society Transactions 952 indeed represented aberrant insertion whereby a local disruption of topology occurred in a fraction of the molecules containing the glycosylation marker in cytoplasmic domains [9]. Nothing is known at present about the tertiary structure of the glucose transporters. We proposed the simplistic model that five amphipathic helices of G L U T l cluster together in the membrane to form an aqueous compartment through which sugars traverse the fatty acyl core of the lipid bilayer [5,10]. Like most membrane proteins, the purified red cell transporter has proved to be recalcitrant to crystallization, and our knowledge of the structure/function relationships of this molecule is limited to what has been revealed through mutagenesis studies. Our initial mutagenesis experiments [ 111 focused on the six tryptophan residues of GLUT1, because it was believed that cytochalasin B, an inhibitor of facilitative glucose transport that binds to G L U T l in the inward-facing conformation, could be covalently bound to the transporter after photoactivation via one or more tryptophan residues. Two of the six tryptophan residues of G L U T l were shown to be critical for full transport function. Substitutions at Trp"' severely reduced the intrinsic activity of G L U T l , whereas substitutions at Trp3XX resulted in a more modest reduction in intrinsic transport activity. Glycine or leucine substitutions at the latter site also caused a rightward shift in a cytochalasin B transport inhibition curve, suggesting that Trp"' is involved in stabilizing the equilibrium binding of cytochalasin B to the transporter. It has also been suggested, on the basis of intrinsic fluorescence experiments conducted on purified erythrocyte transporter, that Trp38Xforms part of a dynamic segment of G L U T l that moves from a position accessible to the aqueous solvent to a solventinaccessible position after substrate binding [ 121. T h e requirement for conformational flexibility in helix 10 is also suggested by the presence of three prolines in this helix, one of which (Pro3") has been shown by mutagenesis studies to be required for full transport activity and for binding of the exofacial ligand 2-N-4-( l-azi2,2,2-trifluoroethyl) benzyl- 1,3-bis- (u-mannos4-yloxy)-2-propylamine (ATP-BMPA) [ 131. Binding of ligands that have a higher affinity for the inward-facing conformation of G L U T l than for the outward-facing conformation, such as cytochalasin B, was not affected by substitutions at We suggested that amide and hydroxy amino Volume 25 acid side chains within the putative amphipathic helices 3, 5, 7, 8 and 11 might provide hydrogenbond donors and acceptors to glucose and thus form the substrate-binding sites [5]. We tested five such residues (Asn'"", Gln"", Gln2"", TyrZx2, TyrZx3)that are conserved in all five G L U T proteins, reasoning that a residue that participates in the formation of a substrate-binding site must be conserved in all members of the G L U T family that transport glucose [14]. Amino acid substitutions at four of the five sites, Asn""' being the exception, reduced intrinsic trapsport activity normalized to the quantity of protein expressed in the oocyte plasma membrane. However, the effect at Gln'"', which resides within transmembrane helix 5, was particularly striking in that even the conservative substitution of asparagine for glutamine reduced intrinsic transport activity by an order of magnitude. This substitution also reduced the apparent affinity of the non-transported glucose analogue, ethylidene glucose, for the transporter by 18-fold. Interestingly, however, the K, for zero trans influx of 2-deoxyglucose was minimally changed by this mutation, whereas the catalytic turnover was decreased by 7.5-fold. These data infer that G1nl6' forms part of the exofacial glucose-binding site and that the asparagine substitution alters the specificity of sugar binding at this site. In addition, the reduced catalytic turnover suggests that substitutions at this position also affect the rate of a conformation change of the carrier involved in net glucose influx. This result is surprising in that the deletion of only a single methylene group from a glutamine side chain has such drastic consequences for transporter function. These observations constituted the first evidence that a residue within the N-terminal half of G L U T l participates in substrate binding. T h e only other residue identified as participating in exofacial substrate binding is GlnZx2, which resides in transmembrane helix 7 [15]. These data on substrate-binding residues are thus consistent with our original suggestion that amideand hydroxy-containing amino acid residues within amphipathic helices may comprise the substrate-binding site(s) [S]. A particularly interesting mutation from a structural as well as a medical standpoint was discovered in the GLUT2 gene of a patient with type-2 diabetes [16]. T h e patient was heterozygous for the Val'97+Ile'y7 mutation, and it is unclear whether this gene defect contributed to the development of diabetes in the patient. How- Structure, Function and Regulation of Glucose Transporters ever, the mutation completely abolished intrinsic transport activity of GLUT2 expressed in Xenopus oocytes. T h e same mutation at the equivalent conserved residue of G L U T l ( ~ a l '-~~' l e ' ~ ' )also abolished transporter activity in oocytes. What is so striking about this observation is that such a subtle structural change, again involving the difference of only a single methylene group at a single residue, has such a dramatic effect on the function of the transporter. This observation also appears somewhat puzzling at first, since Val'"' lies within transmembrane helix 5 , and it is unclear what specific role a valine residue could play in the transport of a hydrophilic molecule such as glucose, particularly in the hydrophobic environment of the lipid bilayer where hydrophobic interactions with the valine side chain cannot contribute to stable inter- or intra-molecular interactions. Our observations with G1nl6' may provide an important clue as to why the Va116'+Ile'b' mutation has such a dramatic functional impact despite the caveats mentioned above. Va1l6' lies approximately one helical turn distant from Gln'", which we know comprises part of the exofacial substrate-binding site and therefore must face the hypothetical aqueous channel in the bilayer formed by the amphipathic transmembrane helices. Thus Val'"' also presumably faces the aqueous channel and also lies very close to the substrate-binding site. T h e extra methylene group at position 165 may be sufficient to impede hydrogen-bond formation between the glucose molecule and the amide group of GlnI6'. Preliminary data from our laboratory support this hypothesis in that small amino acid side chains are better tolerated at position 165 than large side chains. Biosynthesis of GLUT I Perhaps even less is known about the biosynthesis of multispanning membrane proteins than about their structure. G L U T l was one of the first multispanning proteins the biosynthesis of which was studied using synthetic mRNA transcripts translated in an in vitro translation/ translocation system. A very surprising result was that G L U T l could insert post-translationally into microsomes in a process that required phosphoanhydride bond cleavage, and that G L U T l contained at least two functional internal signal sequences [ 7,171. This constituted the first evidence that energy in the form of a nucleoside triphosphate bond was required for the insertion of proteins into or across the endoplasmic reticulum membrane (ER). In addition, these observations contradicted the prevailing theory that insertion of proteins into or across the ER was strictly a co-translational process driven by polypeptide chain elongation. Our data suggest that large domains of membrane proteins may, at least in some instances, be synthesized before their insertion into the membrane. However, the detailed mechanism by which G L U T l or any other polytopic membrane protein inserts into the ER has yet to be elucidated. Once G L U T l is inserted into the ER, in most cells it appears to follow a constitutive route to the plasma membrane, where it contributes to basal glucose uptake. We observed that G L U T l exhibits a relatively rapid transit time through the ER in both Xenopus oocytes and 3T3L1 adipocytes (1 h and 5 min respectively [ 181; transit times through the secretory pathway are in general much slower in oocytes than in mammalian cells). GLUT4, on the other hand, which is approx. 65% identical in sequence with GLUT1, exhibits much slower ER-to-Golgi transit times in both cell types (24 h and 20 min respectively). Because the ER-to-Golgi transit time usually reflects a rate-limiting step in the folding of membrane proteins, we investigated the structural basis for the large difference in this kinetic parameter between G L U T l and GLUT4 by analysis of the biosynthesis of chimaeric G L U T l /GLUT4 molecules expressed in Xenopus oocytes. Unexpectedly, the difference in transit times was localized to discrete structural domains in G L U T l and GLUT4. T h e glycosylated exofacial loop and the C-terminal cytoplasmic tail of both G L U T l and GLUT4 largely determined their transit time behaviour. These observations suggest that these structural domains participate in a rate-limiting step in the folding of the glucose transporter molecules. Conclusions Progress is being made towards understanding the structure of membrane transporters and how that structure is generated during and after the membrane insertion process. T h e G L U T l glucose transporter has served as a useful model to study these processes. However, major progress in the understanding of G L U T l biosynthesis will ultimately require detailed structural information at atomic resolution obtained using high-resolution X-ray crystallography or other spectroscopic methods, a goal I997 953 Biochemical Society Transactions 954 that appears to be technically unfeasible at this time. In the meantime, however, valuable bits and pieces of information can be obtained using the cruder tools of site-directed mutagenesis in conjunction with kinetic and biochemical analyses. Work in the author's laboratory is supported in part by grants from the National Institutes of Health and the American Diabetes Association. Mueckler, M. (1994) Eur. J.Biochem. 219, 713-725 Baldwin, S. A. (1993) Biochirn. Biophys. Acta 1154, 17-49 Mueckler, M. (1992) Curr. Opin. Nephrol. Hypertens. 1, 12-20 Kasahara, M. and Hinkle, P. C. (1977) J. Biol. Chem. 252, 7384-7390 Mueckler, M., Caruso, C., Baldwin, S. A., Panico, M., Blench, I., Morris, €1. R., Allard, W. J., Lienhard, G. E. and Lodish, II. F. (1985) Science 229,941 -945 Widdas, W. F. (1988) Biochim. Biophys. Acta 947, 385-404 Mueckler, M. and Lodish, €1. F. (1986) Nature (London) 322,549-552 Marger, M. D. and Saier, M. J. R. (1993) Trends Biochem. Sci. 18, 13-20 Hresko, R. C., Kruse, M., Strube, M. and Mueckler, M. (1994) J. Biol. Chem. 269, 20482-20488 10 Mueckler, M. (1989) in Red Blood Cell Membranes (Agre, P. and Parker, J. C., eds.), pp. 31-45, Marcel Dekker, New York 11 Garcia, J. C., Strube, M., Leingang, K., Keller, K. and Mueckler, M. M. (1992) J. Biol. Chem. 267, 7770-7776 12 Pawagi, A. B. and Deber, C. M. (1990) Biochemistry 29,950-955 13 Tamori, Y., Hashirarnoto, M., Clark, A. E., Mori, H., Muraoka, A., Kadowaki, T., Holman, G. I>. and Kasuga, M. (1994) J. Biol. Chem. 269, 2982-2986 14 Mueckler, M., Weng, W. and Kruse, M. (1994) J. Biol. Chem. 269,20533-20538 15 Hashiramoto, M., Kadowaki, T., Clark, A. E., Muraoka, A., Momomura, J., Sakura, H., 'robe, K., Akanuma, Y., Yazaki, Y., Holman, G. D. et al. (1992) J.Biol. Chem. 267, 17502-17507 16 Mueckler, M., Kruse, M., Strube, M., Riggs, A. C., Chiu, K. C. and Permutt, M. A. (1994) J. R i d . Chem. 269, 17765-17667 17 Mueckler, M. and I,odish, H.F. (1986) Cell 44, 629-637 18 Hresko, R. C., Murata, H., Marshall, B. A. and Mueckler, M. (1994) J. Biol. Chem. 269, 32110-32119 Received 7 March 1997 Regulation of GLUT1 in response to cellular stress S. A. Baldwin*§, L. F. Barrost, M. Griffiths*, J. Ingram*, E. C. Robbins*, A. J. Streets* and J. SaklatvalaS *Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9]T, U.K., tDepartamento de Medicina Experimental, Facultad de Medicina, Universidad de Chile, lndependencia 1027, Casilla 70058, Santiago 9, Chile, and $The Matilda and Terence Kennedy Institute of Rheumatology, I Aspenlea Road, Hammersmith, London W6 8LH, U.K. A characteristic feature of the early phase of the response of mammalian tissues to metabolic stresses such as hypoxia, ischaemia, osmotic stress, heat shock, viral infection or exposure to metabolic poisons is an increase in the rate of cellular glucose uptake [ 1-31. T h e increase can be large (up to 12-fold in Clone 9 cells exposed to azide [4]) and rapid [ t I l 2 <10 min for Clone 9 Abbreviations used: AICAR, 5-arninoimidazole-4carboxamide ribonucleoside; AMPK, AMP-activated protein kinase; JNK, C-terminal Jun kinase; MAP kinase, mitogen-activated protein kinase; PI 3-kinase, phosphatidylinositol 3-kinase; SAPK, stress-activated protein kinase. $To whom correspondence should be addressed. Volume 25 cells exposed to 0.4 M sorbitol (L. F. Barros and S. A. Baldwin, unpublished work)]. T h i s is an adaptive response, allowing cells to maintain or regain their ATP levels when energy demand increases or oxidative phosphorylation is impaired, allowing the execution of energyrequiring defence programmes such as the synthesis of molecular chaperones, and thus promotes cell survival. After the initial phase of the response (up to 2 h), which is independent of protein synthesis, a second phase occurs, involving 'Ynthesis Of a number Of Proteins, the heat shock proteins and the glucose transporter G L U T 1 [4]. Although stress responses have mainly been studied in cultured cells, they are of great physiological importance