Heterotrimeric GTP-Binding Proteins (G Proteins) and ADP-Ribosylation Factor (ARF) Regulate Priming of Endosomal Membranes for Fusion

An in vitro assay that measures endosome fusion was used to characterize the role of guanosine triphosphate (GTP)-binding proteins in endocytosis. Guanosine 5′,3-(thio)triphosphate (GTPγS), a nonhydrolyzable analog of GTP, stimulates the binding of cytosolic factors to the endosomal membrane (priming). GTPγS also enhances vesicle aggregation, 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 proteins are involved in multiple sequential events preceding endosome fusion. 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 Gβγ subunits of G proteins to the in vitro fusion assay resulted in inhibition of priming. In contrast, addition of ARF to the assay enhanced priming. Thus, heterotrimeric G proteins and ARF may regulate endocytosis by mediating the binding of cytosolic factor(s) required for fusion to the endosomal membrane. Taken together, the results show that multiple GTP-binding proteins regulate a series of distinct biochemical events required for endosome fusion.