Rab5, an early acting endosomal GTPase, supports in vitro endosome fusion without GTP hydrolysis.
Endocytosis is regulated by several GTPases including Rab5 and one or more heterotrimeric G proteins. We show here that Rab5, in the GTP gamma S (guanosine 5'-O-(thiotriphosphate))-bound form, fully supports in vitro endosome fusion, indicating that GTP hydrolysis is not required, whereas Rab5:S34N and Rab5:N133I, mutants unable to bind GTP, are potent inhibitors of endosome fusion. Double mutants (Rab5:S34N/delta C4 and Rab5:N133I/delta C4) lacking the C-terminal prenylation site were inactive, indicating that prenylation is required. Endosomes became resistant to the inhibitory effects of Rab5:S34N by preincubating the vesicles with cytosol prior to the addition of the inhibitor. The acquisition of resistance to Rab5:S34N was more rapid than to N-ethylmaleimide, indicating that Rab5 mutants are early acting. G beta gamma subunits of heterotrimeric G proteins block endosome fusion. However the effect of G beta gamma was abrogated by Rab5-GTP gamma S, indicating that a heterotrimeric G protein may operate upstream of Rab5.
Barbieri
M A
MA
Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, Missouri 63110.
Li
G
G
Colombo
M I
MI
Stahl
P D
PD
D004705:Endocytosis; D011992:Endosomes / Q000201:enzymology*; D019204:GTP-Binding Proteins / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D066298:In Vitro Techniques; D008264:Macrophages / Q000201:enzymology; D008561:Membrane Fusion; D011994:Recombinant Proteins; D020696:rab5 GTP-Binding Proteins
D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins