Stop-Flow Analysis of Cooperative Interactions between GLUT1 Sugar Import and Export Sites

Sultzman, Lisa A.; Carruthers, Anthony

doi:10.1021/bi990130o

Cited by 31 publications

(52 citation statements)

References 24 publications

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“…These findings are compatible with both simple carrier and fixed-site transporter models for transport. Similar studies with nonreduced GLUT1 (120) demonstrate that micromolar levels of exofacial ligands promote one conformational state, whereas higher micromolar levels promote a second, inhibited state. These findings are compatible with only the fixed-site transporter model for transport.…”

Section: Transient Kineticssupporting

confidence: 53%

Will the original glucose transporter isoform please stand up!

Carruthers

DeZutter

Ganguly

et al. 2009

American Journal of Physiology-Endocrinology and Metabolism

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182

190

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Monosaccharides enter cells by slow translipid bilayer diffusion by rapid, protein-mediated, cation-dependent cotransport and by rapid, protein-mediated equilibrative transport. This review addresses protein-mediated, equilibrative glucose transport catalyzed by GLUT1, the first equilibrative glucose transporter to be identified, purified, and cloned. GLUT1 is a polytopic, membranespanning protein that is one of 13 members of the human equilibrative glucose transport protein family. We review GLUT1 catalytic and ligand-binding properties and interpret these behaviors in the context of several putative mechanisms for protein-mediated transport. We conclude that no single model satisfactorily explains GLUT1 behavior. We then review GLUT1 topology, subunit architecture, and oligomeric structure and examine a new model for sugar transport that combines structural and kinetic analyses to satisfactorily reproduce GLUT1 behavior in human erythrocytes. We next review GLUT1 cell biology and the transcriptional and posttranscriptional regulation of GLUT1 expression in the context of development and in response to glucose perturbations and hypoxia in blood-tissue barriers. Emphasis is placed on transgenic GLUT1 overexpression and null mutant model systems, the latter serving as surrogates for the human GLUT1 deficiency syndrome. Finally, we review the role of GLUT1 in the absence or deficiency of a related isoform, GLUT3, toward establishing the physiological significance of coordination between these two isoforms. glucose transport; facilitated diffusion; major facilitator superfamily protein; bloodbrain barrier; placenta; diabetes; glucose transporter 1 deficiency syndrome; development MOST CELLS TRANSPORT SUGARS rapidly down the prevailing concentration gradient into or out of the cell. This equilibrative transport process is mediated by a family of sugar transporters called GLUTs. GLUT1 was the first glucose transporter isoform to be identified, purified (66, 132), and cloned (93) and is one of 13 proteins that comprise the human equilibrative glucose transporter family (63). GLUT1 is a membrane-spanning glycoprotein containing 12 transmembrane domains with a single N-glycosylation site, and its gene is located on chromosome 1 (1p35-31.3) (93). GLUT1 is expressed at the highest levels in the plasma membranes of proliferating cells forming the early developing embryo, in cells forming the blood-tissue barriers, in human erythrocytes and astrocytes, and in cardiac muscle (86). Having a catalytic turnover of ϳ1,200/s (115), glucose transporter 1 (GLUT1) provides an efficient pathway for cellular import and export of glucose. In addition, GLUT1 transports galactose and ascorbic acid (81,107). This review examines the catalytic properties, structure, molecular regulation, and physiology of GLUT1. GLUT1 CATALYTIC PROPERTIES Facilitated DiffusionThe cytoplasm of most cells equilibrates rapidly with nonmetabolizable extracellular sugars. This process is mediated by sugar transport proteins that catalyze unidirectional sugar up...

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Section: Transient Kineticssupporting

confidence: 53%

Will the original glucose transporter isoform please stand up!

Carruthers

DeZutter

Ganguly

et al. 2009

American Journal of Physiology-Endocrinology and Metabolism

Self Cite

182

190

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“…These proteoliposomes are unsealed and simultaneously expose endo-and exofacial GLUT1 domains to exogenous ligands, proteolytic enzymes, and antibodies (26,33). All analyses were performed using untreated GLUT1 or GLUT1 exposed to trypsin or ␣-chymotrypsin for 2 h at 30°C by which time proteolysis is complete as judged by SDS-PAGE or immunoblot analysis (29).…”

Section: Resultsmentioning

confidence: 99%

Analysis of Glucose Transporter Topology and Structural Dynamics

Blodgett

Graybill

Carruthers

2008

Journal of Biological Chemistry

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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 ␣-chymotrypsin, which cuts putative pore-forming amphipathic ␣-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 ␣-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)2 of transport proteins comprises more than 1,000 proteins that mediate passive and secondary active transfer of small molecules across membranes (1). The facilitative glucose transport proteins (GLUT1-12) catalyze monosaccharide uniport in vertebrates (2) and display tissue-specific isoform expression. GLUT1 is expressed in most tissues but is especially abundant in the circulatory system (3) and at blood-tissue barriers such as the blood-brain barrier (4).GLUT1 comprises 492 amino acids; is hydrophobic; contains a single, exofacial N-linked glycosylation site (5); and is predominantly ␣-helical (6). Hydropathy analysis (5), scanning glycosylation mutagenesis (7), proteolysis, antibody binding, and covalent modification studies indicate that GLUT1 contains intracellular N and C termini and 12 transmembrane domain (TM) ␣-helices (8). Amphipathic ␣-helices are proposed to form an aqueous translocation pathway for glucose transport across the plasma membrane (9 -11). 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 (12). The lactose permease (LacY (13)), the glycerol 3-phosphate antiporter (GlpT (14)), ...

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“…Human GLUT1, one of the best-characterized family members, functions as a tetramer (1). Glucose transport is mediated by a fixed-site carrier mechanism, which simultaneously presents cooperative sugar import and export sites (2). Although transport by GLUT1 is intrinsically symmetric (3), kinetic properties for glucose import and export differ and depend, e.g.…”

mentioning

confidence: 99%

“…on redox status or ATP levels (4). Cytochalasin B as an inhibitor of GLUT-mediated transport interacts with the substrate efflux site (2), thus acting as a noncompetitive inhibitor for uptake (5) and competitive inhibitor for release (6,7). Subsequent to uptake, phosphorylation by hexokinases takes place followed by the further glycolytic steps.…”

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confidence: 99%

Imaging of the Dynamics of Glucose Uptake in the Cytosol of COS-7 Cells by Fluorescent Nanosensors

Fehr¹,

Lalonde

Lager

et al. 2003

Journal of Biological Chemistry

279

257

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Glucose homeostasis is a function of glucose supply, transport across the plasma membrane, and metabolism. To monitor glucose dynamics in individual cells, a glucose nanosensor was developed by flanking the Escherichia coli periplasmic glucose/galactose-binding protein with two different green fluorescent protein variants. Upon binding of substrate the FLIPglu-170n sensor showed a concentration-dependent decrease in fluorescence resonance energy transfer between the attached chromophores with a binding affinity for glucose of 170 nM. Fluorescence resonance energy transfer measurements with different sugars indicated a broad selectivity for monosaccharides. An affinity mutant with a K d of ϳ600 M was generated, which showed higher substrate specificity, and thus allowed specific monitoring of reversible glucose dynamics in COS-7 cells in the physiological range. At external glucose concentrations between 0.5 and 10 mM, reflecting typical blood levels, free cytosolic glucose concentrations remained at ϳ50% of external levels. The removal of glucose lead to reduced glucose levels in the cell, demonstrating reversibility and visualizing homeostasis. Glucose levels dropped even in the presence of the transport inhibitor cytochalasin B, indicating rapid metabolism. Consistently, the addition of 2-deoxyglucose, which is not recognized by the sensor, affects glucose uptake and metabolism rates. Within the physiological range, glucose utilization, i.e. hexokinase activity, was not limiting. Furthermore, the results show that in COS-7 cells, cytosolic glucose concentrations can vary over at least two orders of magnitude. The glucose nanosensor provides a novel tool with numerous scientific, medical, and environmental applications.

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Stop-Flow Analysis of Cooperative Interactions between GLUT1 Sugar Import and Export Sites

Cited by 31 publications

References 24 publications

Will the original glucose transporter isoform please stand up!

Will the original glucose transporter isoform please stand up!

Analysis of Glucose Transporter Topology and Structural Dynamics

Imaging of the Dynamics of Glucose Uptake in the Cytosol of COS-7 Cells by Fluorescent Nanosensors

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