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 (GTP gamma S), a nonhydrolyzable analog of GTP, stimulates the binding of cytosolic factors to the endosomal membrane (priming). GTP gamma S 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 G beta gamma 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.
Lenhard
J M
JM
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Colombo
M I
MI
Stahl
P D
PD
D020727:ADP-Ribosylation Factors; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism*; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D010446:Peptide Fragments / Q000494:pharmacology
D010446:Peptide Fragments; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors