ARF-induced lysosomal lysis in vitro.

Abstract

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