Targeting of the "insulin-responsive" glucose transporter (GLUT4) to the regulated secretory pathway in PC12 cells.
Insulin-activated glucose transport depends on the efficient sorting of facilitated hexose transporter isoforms to distinct subcellular locales. GLUT4, the "insulin-responsive" glucose transporter, is sequestered intracellularly, redistributing to the cell surface only in the presence of hormone. To test the hypothesis that the biosynthesis of the insulin-responsive compartment is analogous to the targeting of proteins to the regulated secretory pathway, GLUT4 was expressed in the neuroendocrine cell line, PC12. Localization of the transporter in differentiated PC12 cells by indirect immunofluorescence revealed GLUT4 to be in the perinuclear region and in the distal processes. Although, by immunofluorescence microscopy, GLUT4 co-localized with the endosomal protein transferrin receptor and the small synaptic vesicle (SSV) marker protein synaptophysin, fractionation by velocity gradient centrifugation revealed that GLUT4 was excluded from SSV. Immunoelectron microscopic localization indicated that GLUT4 was indeed targeted to early and late endosomes, but in addition was concentrated in large dense core vesicles (LDCV). This latter observation was confirmed by the following experiments: (a) an antibody directed against GLUT4 immunoadsorbed the LDCV marker protein secretogranin, as assayed by Western blot; (b) approximately 85% of secretogranin metabolically labeled with 35S-labeled sulfate and allowed to progress into secretory vesicles was coadsorbed by an antibody directed against GLUT4; and (c) GLUT4 was readily detected in LDCV purified by ultracentrifugation. These data suggest that GLUT4 is specifically sorted to a specialized secretory compartment in PC12 cells.
Hudson
A W
AW
Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Massachusetts 02115.
Fingar
D C
DC
Seidner
G A
GA
Griffiths
G
G
Burke
B
B
Birnbaum
M J
MJ
D000818:Animals; D002451:Cell Compartmentation; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry; D002864:Chromogranins; D051275:Glucose Transporter Type 4; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D009004:Monosaccharide Transport Proteins / Q000032:analysis / Q000378:metabolism*; D009124:Muscle Proteins; D016501:Neurites / Q000737:chemistry; D015388:Organelles / Q000737:chemistry*; D016716:PC12 Cells; D011506:Proteins / Q000032:analysis; D051381:Rats; D013572:Synaptic Vesicles / Q000737:chemistry; D016708:Synaptophysin / Q000032:analysis; D014162:Transfection
D002864:Chromogranins; D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011506:Proteins; C495694:Slc2a4 protein, rat; D016708:Synaptophysin