GLUT4 IN CULTURED SKELETAL MYOTUBES IS SEGREGATED FROM THE TRANSFERRIN RECEPTOR AND STORED IN VESICLES ASSOCIATED WITH THE TGN

There is little consensus on the nature of the storage compartment of the glucose transporter GLUT4, in non-stimulated cells of muscle and f at, More specifically, it is not known whether GLUT4 is localized to u nique, specialized intracellular storage vesicles, or to vesicles that are part of the constitutive endosomal-lysosomal pathway, To address this question, we have investigated the localization of the endogenous GLUT4 in non-stimulated skeletal myotubes from the cell line C2, by i mmunofluorescence and immunoelectron microscopy, We have used a panel of antibodies to markers of the Golgi complex (alpha mannosidase II an d giantin), of the trans-Golgi network (TGN38), of lysosomes (Igp110), and of early and late endosomes (transferrin receptor and mannose-6-p hosphate receptor, respectively), to define the position of their subc ellular compartments, By immunofluorescence, GLUT4 appears concentrate d in the core of the myotubes, It is primarily found around the nuclei , in a pattern suggesting an association with the Golgi complex, which is further supported by colocalization with giantin and by immunogold electron microscopy, GLUT4 appears to be in the trans-most cisternae of the Golgi complex and in vesicles just beyond, i.e. in the structur es that constitute the trans-Golgi network (TGN), In myotubes treated with brefeldin A, the immunofluorescence pattern of GLUT4 is modified, but it differs from both Golgi complex markers and TGN38, Instead, it resembles the pattern of the transferrin receptor, which forms long t ubules, In untreated cells, double staining for GLUT4 and transferrin receptor by immunofluorescence shows similar but distinct patterns, Im munoelectron microscopy localizes transferrin receptor, detected by im munoperoxidase, to large vesicles, presumably endosomes, very close to the GLUT4-containing tubulovesicular elements, In brefeldin A-treated cells, a network of tubules of similar to 70 nm diameter, studded wit h varicosities, stains for both GLUT4 and transferrin receptor, sugges ting that brefeldin A has caused fusion of the transferrin receptor an d GLUT4-containing compartments, The results suggest that GLUT4 storag e vesicles constitute a specialized compartment that is either a subse t of the TGN, or is very closely linked to it, The link between GLUT4 vesicles and transferrin receptor containing endosomes, as revealed by brefeldin A, may be important for GLUT4 translocation in response to muscle stimulation.