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:...

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Bibliographic Details
Published in:The Journal of biological chemistry 1994-07, Vol.269 (29), p.18720-18722
Main Authors: BARBIERI, M. A, GUANGPU LI, COLOMBO, M. I, STAHL, P. D
Format: Article
Language:English
Subjects:
Biological and medical sciences
Cell physiology
Endocytosis
Endosomes - enzymology
Fundamental and applied biological sciences. Psychology
GTP-Binding Proteins - physiology
Guanosine Triphosphate - metabolism
In Vitro Techniques
Macrophages - enzymology
Membrane Fusion
Molecular and cellular biology
rab5 GTP-Binding Proteins
Recombinant Proteins
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1016/s0021-9258(17)32224-x