In vitro reconstituted Dictyostelium discoideum early endosome fusion is regulated by Rab7 but proceeds in the absence of ATP-Mg2+ from the bulk solution.

The Journal of Biological Chemistry
O LaurentM Satre

Abstract

We characterized the in vitro fusion of endosomal compartments from Dictyostelium discoideum. Fusion activity was restricted to early compartments, was dependent on cytosolic proteins, and was activated by GTP and guanosine 5'-O(3-thio)triphosphate (GTPgammaS). This stimulation suggests the involvement of a small G protein, which we propose to be Rab7 on the basis of the strong inhibitory effect of anti-Rab7 antibodies. It is noteworthy that in the presence of GTPgammaS, the concentration of ATP-Mg2+ could be reduced to less than 1 nM without loss of fusion activity. Under these conditions, competing residual ATP with adenosine 5'-O-(3-thio)triphosphate-Mg2+ also failed to inhibit endosome fusion. The presence of an ATP-depleting system alone blocked fusion probably because endogenous GTP was removed by coupling through NDP kinase. Moreover, whether ATP was present or not, GTPgammaS-activated fusion was equally sensitive to anti-Rab7 antibodies or N-ethylmaleimide and was restricted to early compartments. These results show that soluble ATP-Mg2+ is not needed for endosome fusion. Since homotypic fusion of endosomes in D. discoideum has been shown to depend on the ATPase N-ethylmaleimide-sensitive factor (Lenhard, J. M., Mayorga...Continue Reading

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Citations

Apr 24, 2009·Molecular and Cellular Biochemistry·Sarah J Annesley, Paul R Fisher
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Nov 7, 1998·The Journal of Biological Chemistry·A JahrausG Griffiths
Nov 24, 2006·The Journal of Biological Chemistry·Chaaya Iyengar RajeManoj Raje
Mar 21, 2001·Biochimica Et Biophysica Acta·M Maniak
Mar 21, 2001·Biochimica Et Biophysica Acta·A Rupper, J Cardelli

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