INSULIN-DEPENDENT PROTEIN TRAFFICKING IN SKELETAL-MUSCLE CELLS

We have established a simple procedure for the separation of intracell ular pool(s) of glucose transporter isoform GLUT-4-containing vesicles from the surface sarcolemma and T tubule membranes of rat skeletal my ocytes. This procedure enabled us to immunopurify intracellular GLUT-4 -containing vesicles and to demonstrate that 20-30% of the receptors f or insulin-like growth factor II/mannose B-phosphate and transferrin a re colocalized with GLUT-4 in the same vesicles. Using our new fractio nation procedure as well as cell surface biotinylation, we have shown that these receptors are translocated from their intracellular compart ment(s) to the cell surface along with GLUT-4 after insulin stimulatio n in vivo. Denervation causes a considerable downregulation of GLUT-4 protein in skeletal muscle but does not affect the level of expression of other known component proteins of the corresponding vesicles. More over, the sedimentation coefficient of these vesicles remains unchange d by denervation. We suggest that the normal level of GLUT-4 expressio n is not necessary for the structural organization and insulin-sensiti ve translocation of its cognate intracellular compartment.