Analysis of Glucose Transporter Topology and Structural Dynamics.
UMass Chan Affiliations
Department of Biochemistry and Molecular PharmacologyGraduate School of Biomedical Sciences
Document Type
Journal ArticlePublication Date
2008-12-22Keywords
Biological TransportErythrocytes
Membrane Transport Proteins
Glucose Transporter Type 1
Membrane transport; erythrocytes; GLUT1
Biochemistry, Biophysics, and Structural Biology
Metadata
Show full item recordAbstract
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.Source
J Biol Chem. 2008 Dec 26;283(52):36416-24. Epub 2008 Nov 3. Link to article on publisher's siteDOI
10.1074/jbc.M804802200Permanent Link to this Item
http://hdl.handle.net/20.500.14038/26121PubMed ID
18981181Related Resources
Link to article in PubMedae974a485f413a2113503eed53cd6c53
10.1074/jbc.M804802200
Scopus Count
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