In-Vitro Reconstitution of Phagosome-Endosome Fusion - Evidence for Regulation by Heterotrimeric Gtpases

BERON, W; COLOMBO, MI; MAYORGA, LS; STAHL, PD

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

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Lugar: New York, NY 10159-0945, USA; Año: 1995 vol. 317 p. 337 - 342

0003-9861

We have assessed the role of heterotrimeric GTPases on in vitro fusion of phagosomes and endosomes. Highly purified phagosomes were found to contain G alpha s, G alpha i1, G alpha i2, G alpha i3, and G beta subunits of heterotrimeric GTP-binding proteins. A functional role for G proteins was established using an in vitro phagosome-endosome fusion assay. First, addition of AlF4- and purified G beta gamma subunits to the in vitro assay blocked fusion, indicating that heterotrimeric G proteins may play a role, either direct or indirect, in phagosome maturation. Second, a striking inhibitory effect was observed when the vesicles were incubated with peptides that preferentially activate G alpha s. A similar effect on phagosome-endosome fusion was observed with cholera toxin, a reagent known to activate G alpha s. Our results suggest that one or more heterotrimeric G proteins, including Gs, mediate and/or regulate phagosome-endosome fusion.