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 G alpha s, G alpha i1, G alpha i2, G alpha i3, and G beta sub units of heterotrimeric GTP-binding proteins. A functional role for G proteins was established using an in vitro phagosome-endosome fusion a ssay, First, addition of AlF4- and purified G beta gamma subunits to t he in vitro assay blocked fusion, indicating that heterotrimeric G pro teins may play a role, either direct or indirect, in phagosome maturat ion. Second, a striking inhibitory effect was observed when the vesicl es were incubated with peptides that preferentially activate G alpha s . A similar effect on phagosome-endosome fusion was observed with chol era 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. (C) 1995 Academic Press, Inc.