INHIBITION OF ENDOCYTIC TRANSPORT BY ALUMINUM FLUORIDE IMPLICATES GTPASES AS REGULATORS OF ENDOCYTOSIS

It is now well established that GTP-binding proteins are important reg ulators of vesicular transport. Recent work has shown that multiple GT Pases (both monomeric and heterotrimeric) are required for trafficking . In the present study we have used aluminum fluoride (AIF), a reagent that activates trimeric G proteins, as a tool to study the involvemen t of this family of GTPases in the regulation of endocytosis in intact cells. Our results indicate that AIF inhibits fusion of early endosom es with an intracellular proteolytic compartment. Using the mixing of sequentially internalized ligands as a measure of endocytosis, we foun d that AIF inhibited endocytic transport as assessed by both biochemic al and morphological methods. Taken together these results suggest tha t AIF affects membrane fusion, a common step in vesicular transport. T o further examine the effects of AIF we tested this compound in a cell -free assay that reconstitutes fusion among endosomes. AIF affected en dosomal fusion in a different way than did GTPgammaS, an agent that ac tivates both trimeric and small GTPases. Our results suggest that the coordinated activation of both classes of GTPases are required for eff icient endocytic transport.