Molecular Determinants of Sugar Transport Regulation by ATP ² Kara B. Levine, Erin K. Cloherty, Stephanie Hamill, and Anthony Carruthers* Department of Biochemistry and Molecular Pharmacology, Lazare Research Building, UniVersity of Massachusetts Medical School, 364 Plantation Street, Worcester, Massachusetts 01605 ReceiVed March 29, 2002; ReVised Manuscript ReceiVed July 11, 2002 ABSTRACT: Intracellular ATP inhibits human erythrocyte net sugar transport by binding cooperatively to the glucose transport protein (GluT1). ATP binding produces altered transporter affinity for substrate and promotes substrate occlusion within a post-translocation vestibule formed by GluT1 cytosolic domains. The accompanying paper (Cloherty, E. K., Levine, K. B., Graybill, C., and Carruthers, A. (2002) Biochemistry 41, 12639-12651) demonstrates that reduced intracellular pH promotes high-affinity ATP binding to GluT1 but inhibits ATP-modulation of GluT1-mediated sugar transport. The present study explores the role of GluT1 residues 326-343 (a proposed GluT1 ATP-binding site subdomain) in GluT1 ATP binding by using alanine scanning mutagenesis. Cos-7 and HEK cells were transfected with a cDNA encoding full-length human GluT1 terminating in a carboxyl-terminal hemagglutinin (HA)-His 6 epitope. The transporter (GluT1.HA.H 6 ) is expressed at the surface of both cell-types and is catalytically active. In HEK cells, both parental GluT1- and GluT1.HA.H 6 -mediated sugar transport are acutely sensitive to cellular metabolic inhibition. Isolated, detergent-solubilized GluT1.HA.H 6 is photolabeled by [γ- 32 P]- azidoATP in an ATP-protectable manner. Alanine substitution of E 329 or G 332 /R 333 /R 334 enhances GluT1.HA.H 6 [γ- 32 P]azidoATP photoincorporation but blocks acute modulation of net sugar transport by cellular metabolic inhibition. These actions resemble those of reduced pH on ATP binding to and modulation of red cell GluT1. It is proposed that cooperative nucleotide binding to GluT1 and nucleotide modulation of GluT1-mediated sugar transport are regulated by a proton-sensitive saltbridge (Glu 329 -Arg 333/334 ). Human red blood cell sugar transport is mediated by the integral membrane protein GluT1 1 (1-3). Net sugar transport in human red blood cells is inhibited by intracellular ATP (4). ATP interacts allosterically with GluT1 to alter the tertiary structure of the GluT1 carboxyl terminus (5) and to promote substrate occlusion within a post-translocation pathway vestibule formed by GluT1 cytoplasmic domains (6). Non-hydrolyzable ATP analogues can substitute for ATP in GluT1 regulation, but intracellular AMP and ADP are unable to directly modulate transport. Rather, these nucle- otides compete with ATP for binding to the glucose transporter and thereby competitively inhibit ATP-dependent sugar transport inhibition (5,7). When exposed to extracel- lular reductant, the red cell hexose transfer complex (a GluT1 tetramer) collapses to dimeric GluT1 (8), the cytosolic substrate occlusion vestibule opens (6), GluT1 ATP binding is severely diminished (9, 10), and GluT1-mediated sugar transport is depressed and becomes unresponsive to intra- cellular ATP (6, 8, 11). Reduced intracellular pH enhances ATP binding to red cell GluT1 but, paradoxically, blocks modulation of sugar transport by physiologic intracellular ATP levels (9, 10). ATP binding to GluT1 is a positively cooperative process, and reduced pH converts the low affinity, unliganded carrier into a high-affinity ATP-binding complex (10). We have proposed that the GluT1 cytosolic substrate occlusion vestibule opens when all 4 GluT1 subunits of the sugar transporter complex are liganded by ATP at low pH (10). Sensitivity to H + over the concentration range 10 nM-10 µM suggests that GluT1 histidine side-chain protonation plays a role in modulating sensitivity to and/or binding of ATP. GluT1 contains 5 histidine residues of which four (H 160, 239, 337, and 484) are located within cytoplasmic domains. Exofacial His 50 is an unlikely candidate since red cell sugar transport is insensitive to altered extracellular pH (12). Human erythrocyte GluT1 contains three domains that share homology with human adenylate kinase nucleotide binding domains (5, 13). The GluT1 signature Walker A motif (residues 111-117) lies in an exofacial GluT1 domain (3, 14) and cannot be directly involved in nucleotide binding because ATP acts at an intracellular site on the glucose transporter (9). Adenylate kinase homology domain 2 (residues 225-229) lies in the central cytoplasmic loop of GluT1, and adenylate kinase homology domain 3 (AKHD3; GluT1 residues 332-343) is presented on GluT1 putative ² This work was supported by NIH Grant DK 36081. * To whom correspondence should be addressed. Voice: 508 856 5570. FAX: 508 856 6464. E-mail:anthony.carruthers@umassmed.edu. 1 Abbreviations: GluT1, human erythrocyte glucose transport protein; 2DOG, 2-deoxy-D-glucose; 3MG, 3-O-methyl-R,D-glucopyranoside; C-Ab, rabbit polyclonal antiserum raised against a synthetic peptide comprised of GluT1 residues 480-492; CCB, cytochalasin B; CCD, cytochalasin D; ∂-Ab, sheep polyclonal antiserum raised against native, nonreduced GluT1; EDTA, ethylenediaminetetraacetic acid; FCCP, carbonylcyanide-p-trifluoromethoxyphenyhydrazone; HA-Ab, mouse monoclonal antibody directed against the hemagglutinin sequence LYPYNVPNYA; HEPES, (N-[2-hydroxyethyl]piperazine-N′-[2-ethane- sulfonic acid]); Ni-NTA, nickel-nitrilotriacetic acid; RBC, red blood cell; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electro- phoresis; Tris-HCl, tris(hydroxymethyl)aminomethane. 12629 Biochemistry 2002, 41, 12629-12638 10.1021/bi0258997 CCC: $22.00 © 2002 American Chemical Society Published on Web 09/26/2002