EXOFACIAL EPITOPE-TAGGED GLUCOSE-TRANSPORTER CHIMERAS REVEAL COOH-TERMINAL SEQUENCES GOVERNING CELLULAR-LOCALIZATION

The insulin-regulated adipocyte/skeletal muscle glucose transporter (G LUT4) displays a characteristic steady-state intracellular localizatio n under basal conditions, whereas the erythrocyte/brain transporter is oform (GLUT1) distributes mostly to the cell surface. To identify poss ible structural elements in these transporter proteins that determine their cellular localization, GLUT1/GLUT4 chimera cDNA constructs that contain the hemagglutinin epitope YPYDVPDYA (HA) in their major exofac ial loops were engineered. Binding of monoclonal anti-HA antibody to n on-permeabilized COS-7 cells expressing HA-tagged transporter chimeras revealed that expression of transporters on the cell surface was stro ngly influenced by their cytoplasmic COOH-terminal domain. This method also revealed a less marked, but significant effect on cellular local ization of amino acid residues between transporter exofacial and middl e loops. The subcellular distribution of expressed chimeras was confir med by immunofluorescence microscopy of permeabilized COS-7 cells. Thu s, HA-tagged native GLUT4 was concentrated in the perinuclear region, whereas a chimera containing the COOH-terminal 29 residues of GLUT1 su bstituted onto GLUT4 distributed to the plasma membrane, as did native GLUT1. Furthermore, a chimera composed of GLUT1 with a GLUT4 COOH-ter minal 30-residue substitution exhibited a predominantly intracellular localization. Similar data was obtained in CHO cells stably expressing these chimeras. Taken together, these results define the unique COOH- terminal cytoplasmic sequences of the GLUT1 and GLUT4 glucose transpor ters as important determinants of cellular localization in COS-7 and C HO cells.