title: Differential localization of glucose transporter isoforms in non-polarized mammalian cells: distribution of GLUT1 but not GLUT3 to detergent-resistant membrane domains.
abstract: The hexose transporter family, which mediates a facilitated uptake in mammalian cells, consists of more than 10 members containing 12 membrane-spanning segments with a single N-glycosylation site. However, it remains unknown how these isoforms are functionally organized in the membrane domains. In this report, we describe a differential distribution of the glucose transporter isoforms GLUT1 and GLUT3 to detergent-resistant membrane domains (DRMs) in non-polarized mammalian cells. Whereas more than 80% of cellular proteins containing GLUT3 in HeLa cell lines was solubilized by a non-ionic detergent (either Triton X-100 or Lubrol WX) at 4 degrees C, GLUT1 remained insoluble together with the DRM-associated proteins, such as caveolin-1 and intestinal alkaline phosphatase (IAP). These DRM-associated proteins and the ganglioside GM1 were shown to float to the upper fractions when Triton X-100-solubilized cell extracts were centrifuged on a density gradient. In contrast, GLUT3 as well as most soluble proteins remained in the lower layers. Furthermore, perturbations of DRMs due to depletion of cholesterol by methyl-beta-cyclodextrin (m beta CD) rendered GLUT1 soluble in Triton X-100. Immunostaining patterns for these isoforms detected by confocal laser scanning microscopy in a living cell were also distinctive. These results suggest that in non-polarized mammalian cells, GLUT1 can be organized into a raft-like DRM domain but GLUT3 may distribute to fluid membrane domains. This differential distribution may occur irrespective of the N-glycosylation state or cell type.
authors.affiliation: Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Sakyo
authors.forename: Takayuki
authors.initials: T
authors.lastname: Kitagawa
mesh_terms: D015153:Blotting, Western; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique; D051272:Glucose Transporter Type 1; D051274:Glucose Transporter Type 3; D006367:HeLa Cells; D006801:Humans; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins; D017830:Octoxynol / Q000494:pharmacology; D012995:Solubility
chemical_list: D003902:Detergents; D051272:Glucose Transporter Type 1; D051274:Glucose Transporter Type 3; D009004:Monosaccharide Transport Proteins; D009419:Nerve Tissue Proteins; C495677:SLC2A1 protein, human; C495687:SLC2A3 protein, human; D017830:Octoxynol