title: Cysteine scanning mutagenesis of helices 2 and 7 in GLUT1 identifies an exofacial cleft in both transmembrane segments.
abstract: Cysteine scanning mutagenesis in conjunction with site-directed chemical modification of sulfhydryl groups by p-chloromercuribenzenesulfonate (pCMBS) or N-ethylmaleimide (NEM) was applied to putative transmembrane segments (TM) 2 and 7 of the cysteine-less glucose transporter GLUT1. Valid for both helices, the majority of cysteine substitution mutants functioned as active glucose transporters. The residues F72, G75, G76, G79, and S80 within helix 2 and G286 and N288 within helix 7 were irreplaceable because the mutant transporters displayed transport activities that were lower than 10% of Cys-less GLUT1. The indicated cluster of glycine residues within TM 2 is located on one face of the helix and may provide space for a bulky hydrophobic counterpart interacting with another transmembrane segment or lipid side chains. Characteristic for helix 7, three glutamine residues (Q279, Q282, and Q283) played an important role in transport activity of Cys-less GLUT1 because an individual replacement with cysteine reduced their transport rates by about 80%. ParaCMBS-sensitivity scanning of both transmembrane segments detected several membrane-harbored residues to be accessible to the extracellular aqueous solvent. The pCMBS-reactive sulfhydryl groups were located exclusively in the exofacial half of the plasma membrane and, when presented in a helical model, lie along one side of the helices. Taken together, transmembrane segments 2 and 7 form clefts accessible to the extracellular aqueous solvent. The lining residues are however excluded from interaction with intracellular solutes, as justified by microinjection of pCMBS into the cytoplasm of Xenopus oocytes.
authors.affiliation: Institut für Pharmakologie, Freie Universität Berlin, Thielallee 67-73, D-14195 Berlin, FRG, Institut für Molekulare Pharmakologie, Alfred Kowalke Str. 4, D-10315 Berlin, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Olsowski
authors.forename: I
authors.initials: I
authors.lastname: Monden
authors.forename: G
authors.initials: G
authors.lastname: Krause
authors.forename: K
authors.initials: K
authors.lastname: Keller
mesh_terms: D002731:4-Chloromercuribenzenesulfonate / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D003198:Computer Simulation; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism; D005033:Ethylmaleimide / Q000378:metabolism; D051272:Glucose Transporter Type 1; D005973:Glutamine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013439:Sulfhydryl Reagents / Q000378:metabolism
chemical_list: D051272:Glucose Transporter Type 1; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; C495677:SLC2A1 protein, human; D013439:Sulfhydryl Reagents; D007532:Isoleucine; D005973:Glutamine; D002731:4-Chloromercuribenzenesulfonate; D003545:Cysteine; D005033:Ethylmaleimide