title: Sequence determinants of GLUT1 oligomerization: analysis by homology-scanning mutagenesis.
abstract: The human blood-brain barrier glucose transport protein (GLUT1) forms homodimers and homotetramers in detergent micelles and in cell membranes, where the GLUT1 oligomeric state determines GLUT1 transport behavior. GLUT1 and the neuronal glucose transporter GLUT3 do not form heterocomplexes in human embryonic kidney 293 (HEK293) cells as judged by co-immunoprecipitation assays. Using homology-scanning mutagenesis in which GLUT1 domains are substituted with equivalent GLUT3 domains and vice versa, we show that GLUT1 transmembrane helix 9 (TM9) is necessary for optimal association of GLUT1-GLUT3 chimeras with parental GLUT1 in HEK cells. GLUT1 TMs 2, 5, 8, and 11 also contribute to a less abundant heterocomplex. Cell surface GLUT1 and GLUT3 containing GLUT1 TM9 are 4-fold more catalytically active than GLUT3 and GLUT1 containing GLUT3 TM9. GLUT1 and GLUT3 display allosteric transport behavior. Size exclusion chromatography of detergent solubilized, purified GLUT1 resolves GLUT1/lipid/detergent micelles as 6- and 10-nm Stokes radius particles, which correspond to GLUT1 dimers and tetramers, respectively. Studies with GLUTs expressed in and solubilized from HEK cells show that HEK cell GLUT1 resolves as 6- and 10-nm Stokes radius particles, whereas GLUT3 resolves as a 6-nm particle. Substitution of GLUT3 TM9 with GLUT1 TM9 causes chimeric GLUT3 to resolve as 6- and 10-nm Stokes radius particles. Substitution of GLUT1 TM9 with GLUT3 TM9 causes chimeric GLUT1 to resolve as a mixture of 6- and 4-nm particles. We discuss these findings in the context of determinants of GLUT oligomeric structure and transport function.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
authors.forename: Julie K
authors.initials: JK
authors.lastname: De Zutter
authors.forename: Kara B
authors.initials: KB
authors.lastname: Levine
authors.forename: Di
authors.initials: D
authors.lastname: Deng
authors.forename: Anthony
authors.initials: A
authors.lastname: Carruthers
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003847:Deoxyglucose / Q000378:metabolism / Q000493:pharmacokinetics; D003902:Detergents / Q000737:chemistry; D051272:Glucose Transporter Type 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051274:Glucose Transporter Type 3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008823:Micelles; D008969:Molecular Sequence Data; D009154:Mutation; D015202:Protein Engineering; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D003902:Detergents; D051272:Glucose Transporter Type 1; D051274:Glucose Transporter Type 3; D008823:Micelles; D011993:Recombinant Fusion Proteins; C495677:SLC2A1 protein, human; C495687:SLC2A3 protein, human; D003847:Deoxyglucose
keywords: Erythrocyte; Glucose Transport; Membrane Proteins; Membrane Transport; Neurons