Recycling of the insulin-responsive glucose transporter Glut4 regulated by the small GTPase RalA and the exocyst complex.

Endocytic recycling represents a major mechanism for continuous supply of molecules to the plasma membrane. Particularly, outbound trafficking of the recycling endosome (RE) or RE-derived vesicles can be upregulated by cellular signaling, through mobilization of specialized protein complexes acting as transport machineries. Therefore, biochemical and functional characterization of cell signaling molecules that operate multimeric protein complexes in membrane transport provides important insights to signaling-regulated trafficking events. In this chapter, we described biochemical approaches and reporter assays in differentiated adipocytes to determine the activity and function of the small GTPase RalA, which relays upstream insulin signaling to the exocyst complex that targets intracellular vesicles bearing the Glut4 transporter to the plasma membrane. The experimental design outlined in this chapter can be applied to other regulated transport events facilitated by the exocyst complex, as well as other GTPases that operate distinct transport complexes in specific physiological settings.