GTPASE-ACTIVATING PROTEIN-ACTIVITY FOR RAB4 IS ENRICHED IN THE PLASMA-MEMBRANE OF 3T3-L1 ADIPOCYTES - POSSIBLE INVOLVEMENT IN THE REGULATION OF RAB4 SUBCELLULAR-LOCALIZATION

The small guanosine 5'-triphosphate (GTP)ase Rab4 has been suggested t o play a role in insulin-induced GLUT4 translocation. Under insulin st imulation, GLUT4 translocates to the plasma membranes, while Rab4 leav es the GLUT4-containing vesicles and becomes cytosolic. Rab proteins c ycle between a GTP-bound active form and a guanosine 5'-diphosphate (G DP)-bound inactive form. The intrinsic GTPase activity of Rab proteins is low and the interconversion between the two forms is dependent on accessory factors. In the present work: we searched for a GTPase activ ating protein (GAP) for Rab4 in 3T3-L1 adipocytes. We used a glutathio ne-transferase (GST)-Rab4 protein which possesses the properties of a small GTPase (ability to bind GDP and GTP and to hydrolyse GTP) and ca n be isolated in a rapid and efficient way. This GAP activity was obse rved in 3T3-L1 adipocyte lysates, and was able to accelerate the hydro lysis of the [alpha-P-32]GTP bound to GST-Rab4 into [alpha-P-32]GDP. T his activity, tentatively called Rab4-GAP, was also present in 3T3-L1 fibroblasts. The Rab4-GAP activity was present in total membrane fract ions and nearly undetectable in cytosol. Following subcellular fractio nation, Rab4-GAP was found to be enriched in plasma membranes when com pared to internal microsomes. Insulin treatment of the cells had no ef fect on the total Rab4-GAP activity or on its subcellular localization . Taking our results together with the accepted model of Rab cycling i n intracellular traffic, we propose that Rab4-GAP activity plays a rol e in the cycling between the GTP- and GDP-bound forms of Rab4, and thu s possibly in the traffic of GLUT4-containing vesicles.