GLUT4 AND TRANSFERRIN RECEPTOR ARE DIFFERENTIALLY SORTED ALONG THE ENDOCYTIC PATHWAY IN CHO CELLS

The trafficking of GLUT4, a facilitative glucose transporter, is exami ned in transfected CHO cells. In previous work, we expressed GLUT4 in neuroendocrine cells and fibroblasts and found that it was targeted to a population of small vesicles slightly larger than synaptic vesicles (Herman, G.A, F. Bonzelius, A.M. Cieutat, and R.B. Kelly. 1994. Proc. Natl. Acad. Sci. USA. 91:12750-12754.). In this study, we demonstrate that at 37 degrees C, GLUT4-containing small vesicles (GSVs) are dete cted after cell surface radiolabeling of GLUT4 whereas uptake of radio iodinated human transferrin does not show appreciable accumulation wit hin these small vesicles. Immunofluorescence microscopy experiments sh ow that at 37 degrees C, cell surface-labeled GLUT4 as well as transfe rrin is internalized into peripheral and perinuclear structures. At 15 degrees C, endocytosis of GLUT4 continues to occur at a slowed rate, but whereas fluorescently labeled GLUT4 is seen to accumulate within l arge peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37 degrees C af ter accumulating labeled GLUT4 at 15 degrees C results in the reappear ance of GLUT4 in perinuclear structures and GSV reformation. Cytosol a cidification or treatment with hypertonic media containing sucrose pre vents the exit of GLUT4 from peripheral endosomes as well as GSV forma tion, suggesting that coat proteins may be involved in the endocytic t rafficking of GLUT4. In contrast, at 15 degrees C, transferrin continu es to traffic to perinuclear structures and overall labels structures similar in distribution to those observed at 37 degrees C. Furthermore , treatment with hypertonic media has no apparent effect on transferri n trafficking from peripheral endosomes. Double-labeling experiments a fter the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclu de the transferrin receptor (TfR) at both 15 degrees and 37 degrees C, Thus, GLUT4 is sorted differently from the transferrin receptor as ev idenced by the targeting of each protein to distinct early endosomal c ompartments and by the formation of GSVs. These results suggest that t he sorting of GLUT4 from TfR may occur primarily at the level of the p lasma membrane into distinct endosomes and that the organization of th e endocytic system in CHO cells more closely resembles that of neuroen docrine cells than previously appreciated.