ARF-INDUCED LYSOSOMAL LYSIS IN-VITRO

Cytosol treated with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) disintegrated lysosomes in a dose-dependent manner, as detected as th e release of preloaded fluorescein isothiocyanate-dextran. The effect of GTP gamma S was suppressed by GTP or GDP, indicating a role of a GT P binding protein (G-protein) in the lysis [Sai, Y, et al, (1994) Bioc hem. Biophys. Res. Commun. 198, 869-877], Gel filtration of cytosol an d GTP-ligand blotting showed that a small GTP-binding protein particip ated in the lysosomal lysis, We partially purified the G-protein from rat liver cytosol and identified it as ARF1. GTP gamma S-stimulated ly sis was reconstituted with ARF1 purified from bovine brain cytosol or recombinant ARF1, ARF bound to lysosomal membranes depending upon GTP gamma S in a dose-dependent manner, These results suggest that the tra nsfer of ARF from the cytosol to the lysosomal membrane is necessary f or GTP gamma S-stimulated lysis of lysosomes.