UMMS Affiliation

Program in Molecular Medicine; Department of Cell Biology

Publication Date

10-1-1993

Document Type

Article

Subjects

Amino Acid Sequence; Animals; Biological Markers; CHO Cells; *Cell Compartmentation; Cells, Cultured; Cricetinae; Fluorescent Antibody Technique; Glucose Transporter Type 1; Glucose Transporter Type 4; Hemagglutinins; Molecular Sequence Data; Monosaccharide Transport Proteins; *Muscle Proteins; Recombinant Fusion Proteins; Structure-Activity Relationship

Disciplines

Cell Biology

Abstract

The insulin-regulated adipocyte/skeletal muscle glucose transporter (GLUT4) displays a characteristic steady-state intracellular localization under basal conditions, whereas the erythrocyte/brain transporter isoform (GLUT1) distributes mostly to the cell surface. To identify possible 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 exofacial loops were engineered. Binding of monoclonal anti-HA antibody to non-permeabilized COS-7 cells expressing HA-tagged transporter chimeras revealed that expression of transporters on the cell surface was strongly influenced by their cytoplasmic COOH-terminal domain. This method also revealed a less marked, but significant effect on cellular localization of amino acid residues between transporter exofacial and middle loops. The subcellular distribution of expressed chimeras was confirmed by immunofluorescence microscopy of permeabilized COS-7 cells. Thus, HA-tagged native GLUT4 was concentrated in the perinuclear region, whereas a chimera containing the COOH-terminal 29 residues of GLUT1 substituted onto GLUT4 distributed to the plasma membrane, as did native GLUT1. Furthermore, a chimera composed of GLUT1 with a GLUT4 COOH-terminal 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 transporters as important determinants of cellular localization in COS-7 and CHO cells.

Rights and Permissions

Citation: J Cell Biol. 1993 Oct;123(1):127-35.

Related Resources

Link to Article in PubMed

Journal/Book/Conference Title

The Journal of cell biology

PubMed ID

8408193

Included in

Cell Biology Commons

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