ARF-induced lysosomal lysis in vitro.
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.
Arai
K
K
Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920-0934.
Matsuda
T
T
Sai
Y
Y
Ohkuma
S
S
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors