CHARACTERIZATION OF THE INTRACELLULAR GLUT-4 COMPARTMENT

Insulin stimulates glucose transport in muscle and adipose tissue by triggering the translocation of the glucose transporter GLUT-4 from intracellular vesicles to the cell surface. In the present study we have attempted to characterize the intracellular GLUT-4 compartment using vesicle immunoadsorption. Silver staining of this fraction indicates that this compartment contains numerous polypeptides that exhibit a marked change in mobility upon treatment with reducing agents. The polypeptide composition of GLUT-4-containing vesicles isolated from a variety of insulin-sensitive cell types, including heart, adipose tissue, skeletal muscle and 3T3-L1 adipocytes, is similar. In addition, the polypeptide composition of the GLUT-4 compartment isolated from CHO cells transfected with GLUT-4 resembles that observed in insulin-sensitive cells. Two major proteins in this vesicle fraction isolated from all cell types are the transferrin receptor (TfR) and the mannose 6-phosphate/IGF II receptor (MPR). Furthermore, vesicles immunoadsorbed from adipocytes, with antibodies specific for GLUT-4 and the TfR, also show conservation in their overall polypeptide composition. Protein micro sequencing of a major 80 kDa polypeptide enriched in the GLUT-4 compartment isolated from skeletal muscle revealed this protein to be rat transferrin, These data indicate that there is a close relationship between the intracellular GLUT-4 compartment and the endosomal system. Future studies will be required to determine if it is possible to isolate subcompartments within this system to determine if GLUT-4 is targeted to a specialized secretory compartment in insulin-sensitive cells or simply a subdomain within recycling endosomes.