HETEROTRIMERIC GTP-BINDING PROTEINS (G-PROTEINS) AND ADP-RIBOSYLATIONFACTOR (ARF) REGULATE PRIMING OF ENDOSOMAL MEMBRANES FOR FUSION

An in vitro assay that measures endosome fusion was used to characteri ze the role of guanosine triphosphate (GTP)-binding proteins in endocy tosis. Guanosine 5',3-(thio)triphosphate (GTP gamma S), a nonhydrolyza ble analog of GTP, stimulates the binding of cytosolic factors to the endosomal membrane (priming). GTP gamma S also enhances vesicle aggreg ation, resulting in the formation of an intermediate that is resistant to dilution. In this report we demonstrate that priming precedes the appearance of a dilution-resistant intermediate. Thus, GTP-binding pro teins are involved in multiple sequential events preceding endosome fu sion. Both heterotrimeric G proteins (G proteins) and ADP-ribosylation factors (ARFs) are GTP-binding proteins that regulate undefined steps involved in endocytosis. The addition of G beta gamma subunits of G p roteins to the in vitro fusion assay resulted in inhibition of priming . In contrast, addition of ARF to the assay enhanced priming. Thus, he terotrimeric G proteins and ARF may regulate endocytosis by mediating the binding of cytosolic factor(s) required for fusion to the endosoma l membrane. Taken together, the results show that multiple GTP-binding proteins regulate a series of distinct biochemical events required fo r endosome fusion. (C) 1994 Academic Press, Inc.