title: Domains that confer intracellular sequestration of the Glut4 glucose transporter in Xenopus oocytes.
abstract: The Glut4 glucose transporter is poorly functional compared with other glucose transporter isoforms when expressed in Xenopus oocytes. To investigate the molecular basis for this poor functionality, we compared the biosynthesis and targeting of Glut1 and Glut4 in oocytes after microinjection of the corresponding mRNAs. Both Glut1 and Glut4 were present as lower molecular weight endoglycosidase H-sensitive and higher molecular weight endoglycosidase H-resistant. Subcellular fractionation indicated that Glut1 was targeted to the plasma membrane with a 6.6-fold greater efficiency than was Glut4. Confocal immunofluorescence microscopy confirmed the relative enrichment of Glut1 in the plasma membrane and the efficient intracellular sequestration of Glut4. As in mammalian cells, the endoglycosidase H-resistant form of Glut4 was concentrated in low-density intracellular vesicles, whereas Glut1 was distributed in intracellular vesicles of higher average density. The structural basis for the differential localization of Glut1 and Glut4 was investigated by determining the plasma membrane content of a series of chimeric Glut1/Glut4 molecules. These data indicated that two distinct regions of Glut4, encompassing residues 24-132 and the COOH-terminal cytoplasmic tail, confer intracellular sequestration on the chimeric transporter molecules. At least part of the sequestration effect of the more N-terminal domain was due to the incomplete maturation of chimeras containing this region, resulting in the accumulation of lower molecular weight endoglycosidase H-sensitive and endoglycosidase H-resistant forms, whereas the COOH-terminal cytoplasmic tail conferred sequestration of fully glycosylated chimeras in a low-density intracellular membrane compartment.
authors.affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: B A
authors.initials: BA
authors.lastname: Marshall
authors.forename: H
authors.initials: H
authors.lastname: Murata
authors.forename: R C
authors.initials: RC
authors.lastname: Hresko
authors.forename: M
authors.initials: M
authors.lastname: Mueckler
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005260:Female; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D006801:Humans; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D009865:Oocytes / Q000378:metabolism*; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495677:SLC2A1 protein, human; C495692:SLC2A4 protein, human; C495679:Slc2a1 protein, rat; C495694:Slc2a4 protein, rat