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GLUT4 and transferrin receptor are differentially sorted along the endocytic pathway in CHO cells.

Wei ML, Bonzelius F, Scully RM, Kelly RB, Herman GA - J. Cell Biol. (1998)

Bottom Line: Shifting cells to 37 degrees C after accumulating labeled GLUT4 at 15 degrees C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation.Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes.These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, The Hormone Research Institute, University of California, San Francisco, California 94143, USA.

ABSTRACT
The trafficking of GLUT4, a facilitative glucose transporter, is examined 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 detected after cell surface radiolabeling of GLUT4 whereas uptake of radioiodinated human transferrin does not show appreciable accumulation within these small vesicles. Immunofluorescence microscopy experiments show that at 37 degrees C, cell surface-labeled GLUT4 as well as transferrin 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 large peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37 degrees C after accumulating labeled GLUT4 at 15 degrees C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation. Cytosol acidification or treatment with hypertonic media containing sucrose prevents the exit of GLUT4 from peripheral endosomes as well as GSV formation, suggesting that coat proteins may be involved in the endocytic trafficking of GLUT4. In contrast, at 15 degrees C, transferrin continues 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 transferrin trafficking from peripheral endosomes. Double-labeling experiments after the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclude the transferrin receptor (TfR) at both 15 degrees and 37 degrees C. Thus, GLUT4 is sorted differently from the transferrin receptor as evidenced by the targeting of each protein to distinct early endosomal compartments and by the formation of GSVs. These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.

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Double-label immunofluorescence with internalized GLUT4 and transferrin. Cells were incubated  with both Texas red–coupled  transferrin and fluoresceinated 9E10 at 15°C for 2.5 h,  and then either processed  immediately for microscopy  (A–F) or shifted to 37°C for  10 min followed by processing for microscopy (G–I). A,  D, and G, FITC-9E10; B, E,  and H: Texas red–transferrin; and C, F, and I: merged  images. Areas of overlap are  indicated in yellow. Bars:  (A–F) 2 μm; (G–I) 10 μm.
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Figure 5: Double-label immunofluorescence with internalized GLUT4 and transferrin. Cells were incubated with both Texas red–coupled transferrin and fluoresceinated 9E10 at 15°C for 2.5 h, and then either processed immediately for microscopy (A–F) or shifted to 37°C for 10 min followed by processing for microscopy (G–I). A, D, and G, FITC-9E10; B, E, and H: Texas red–transferrin; and C, F, and I: merged images. Areas of overlap are indicated in yellow. Bars: (A–F) 2 μm; (G–I) 10 μm.

Mentions: The large peripheral GLUT4-containing compartment observed at 15°C was further characterized by double- labeling immunofluorescence assays. Cells were incubated at 15°C for 2.5 h with both Texas red–coupled transferrin and fluoresceinated anti-myc antibody. Cells were then either processed immediately (Fig. 5, A–F) for confocal microscopy or shifted to 37°C for 10 min in label-free media (Fig. 5, G–I), and then processed. At 15°C, large peripheral compartments containing GLUT4 but not transferrin were identified. The perinuclear structures containing transferrin were less apparent in these images because of the plane of sectioning, which was chosen to optimize the visualization of the GLUT4 structures. After shifting to 37°C, GLUT4 and TfR did appear to be colocalized to some extent in the perinuclear region, although the possibility of distinct GLUT4- and transferrin-containing compartments in very close apposition is not eliminated. In the periphery, many structures enriched for GLUT4 but not for the TfR were apparent, suggesting that GLUT4 sorts away from the TfR at 37°C as well. This latter pattern (Fig. 5, G–I) is identical to that seen in cells which have been incubated with both labels at 37°C for 30 min, without a prior incubation at 15°C (data not shown).


GLUT4 and transferrin receptor are differentially sorted along the endocytic pathway in CHO cells.

Wei ML, Bonzelius F, Scully RM, Kelly RB, Herman GA - J. Cell Biol. (1998)

Double-label immunofluorescence with internalized GLUT4 and transferrin. Cells were incubated  with both Texas red–coupled  transferrin and fluoresceinated 9E10 at 15°C for 2.5 h,  and then either processed  immediately for microscopy  (A–F) or shifted to 37°C for  10 min followed by processing for microscopy (G–I). A,  D, and G, FITC-9E10; B, E,  and H: Texas red–transferrin; and C, F, and I: merged  images. Areas of overlap are  indicated in yellow. Bars:  (A–F) 2 μm; (G–I) 10 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140164&req=5

Figure 5: Double-label immunofluorescence with internalized GLUT4 and transferrin. Cells were incubated with both Texas red–coupled transferrin and fluoresceinated 9E10 at 15°C for 2.5 h, and then either processed immediately for microscopy (A–F) or shifted to 37°C for 10 min followed by processing for microscopy (G–I). A, D, and G, FITC-9E10; B, E, and H: Texas red–transferrin; and C, F, and I: merged images. Areas of overlap are indicated in yellow. Bars: (A–F) 2 μm; (G–I) 10 μm.
Mentions: The large peripheral GLUT4-containing compartment observed at 15°C was further characterized by double- labeling immunofluorescence assays. Cells were incubated at 15°C for 2.5 h with both Texas red–coupled transferrin and fluoresceinated anti-myc antibody. Cells were then either processed immediately (Fig. 5, A–F) for confocal microscopy or shifted to 37°C for 10 min in label-free media (Fig. 5, G–I), and then processed. At 15°C, large peripheral compartments containing GLUT4 but not transferrin were identified. The perinuclear structures containing transferrin were less apparent in these images because of the plane of sectioning, which was chosen to optimize the visualization of the GLUT4 structures. After shifting to 37°C, GLUT4 and TfR did appear to be colocalized to some extent in the perinuclear region, although the possibility of distinct GLUT4- and transferrin-containing compartments in very close apposition is not eliminated. In the periphery, many structures enriched for GLUT4 but not for the TfR were apparent, suggesting that GLUT4 sorts away from the TfR at 37°C as well. This latter pattern (Fig. 5, G–I) is identical to that seen in cells which have been incubated with both labels at 37°C for 30 min, without a prior incubation at 15°C (data not shown).

Bottom Line: Shifting cells to 37 degrees C after accumulating labeled GLUT4 at 15 degrees C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation.Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes.These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, The Hormone Research Institute, University of California, San Francisco, California 94143, USA.

ABSTRACT
The trafficking of GLUT4, a facilitative glucose transporter, is examined 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 detected after cell surface radiolabeling of GLUT4 whereas uptake of radioiodinated human transferrin does not show appreciable accumulation within these small vesicles. Immunofluorescence microscopy experiments show that at 37 degrees C, cell surface-labeled GLUT4 as well as transferrin 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 large peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37 degrees C after accumulating labeled GLUT4 at 15 degrees C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation. Cytosol acidification or treatment with hypertonic media containing sucrose prevents the exit of GLUT4 from peripheral endosomes as well as GSV formation, suggesting that coat proteins may be involved in the endocytic trafficking of GLUT4. In contrast, at 15 degrees C, transferrin continues 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 transferrin trafficking from peripheral endosomes. Double-labeling experiments after the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclude the transferrin receptor (TfR) at both 15 degrees and 37 degrees C. Thus, GLUT4 is sorted differently from the transferrin receptor as evidenced by the targeting of each protein to distinct early endosomal compartments and by the formation of GSVs. These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.

Show MeSH
Related in: MedlinePlus