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

We have assessed the role of heterotrimeric GTPases on in vitro fusion of phagosomes and endosomes. Highly purified phagosomes were found to contain Gαs, Gαi1, Gαi2, Gαi3, and Gβ 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 AlF−4 and purified Gβγ 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 Gαs. A similar effect on phagosome-endosome fusion was observed with cholera toxin, a reagent known to activate Gαs. Our results suggest that one or more heterotrimeric G proteins, including Gs, mediate and/or regulate phagosome-endosome fusion.