Analysis of glucose transporter topology and structural dynamics.
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 alpha-chymotrypsin, which cuts putative pore-forming amphipathic alpha-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 alpha-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.
Blodgett
David M
DM
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Graybill
Christopher
C
Carruthers
Anthony
A
D000595:Amino Acid Sequence; D001665:Binding Sites; D003571:Cytochalasin B / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D051272:Glucose Transporter Type 1 / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology
D051272:Glucose Transporter Type 1; C495677:SLC2A1 protein, human; D003571:Cytochalasin B; D014357:Trypsin