Association of a 19- and a 21-kDa GTP-binding protein to pancreatic microsomal vesicles is regulated by the intravesicular pH established by a vacuolar-type H(+)-ATPase.
Evidence suggests that certain ras-related small molecular weight GTP-binding proteins (smg-proteins) are involved in intracellular membrane trafficking and vesicle fusion. We have previously shown that intravesicular acidification due to a vacuolar-type H(+)-ATPase, which is Cl- dependent and highly sensitive to the specific inhibitor bafilomycin, enhances GTP-induced fusion of pancreatic microsomal vesicles (Hampe, W., Zimmermann, P., Schulz, I. 1990. FEBS Lett. 271:62-66). This process may involve function of smg-proteins. The present study shows that MgATP (2 mM), but neither MgATP gamma S nor ATP in the absence of Mg2+, increases association of 19- and 21-kDa smg-proteins to the vesicle membrane as monitored by their [ alpha-32P]GTP binding. The affinity of smg-proteins for [ alpha-32P]GTP was not altered by MgATP. Bafilomycin B1 (10(-8) M), the protonophore CCCP (10(-5) M), and replacement of Cl- in the incubation buffer by CH3COO- or NO3- resulted in an almost complete inhibition of the MgATP-dependent association of the 19- and 21-kDa smg-proteins to the vesicle membranes. Furthermore, the MgATP effect on both smg-proteins was found to be due to the intravesicular pH and not to the H+ gradient over the vesicle membrane. We conclude that association of a 19-kDa (immunologically identified as the ADP-ribosylation factor, arf) and a yet unidentified 21-kDa GTP-binding protein to vesicle membranes is regulated by the intravesicular pH established by a vacuolar-type H(+)-ATPase.
Zeuzem
S
S
Max-Planck-Institut für Biophysik, Frankfurt am Main, Germany.
Zimmermann
P
P
Schulz
I
I
D000255:Adenosine Triphosphate / Q000378:metabolism; D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001667:Binding, Competitive; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D003902:Detergents; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018942:Macrolides; D008861:Microsomes / Q000378:metabolism*; D010179:Pancreas / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D051381:Rats; D012492:Salts; D014617:Vacuoles / Q000201:enzymology*
D000477:Alkylating Agents; D000900:Anti-Bacterial Agents; D003902:Detergents; D018942:Macrolides; D012492:Salts; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; C052501:bafilomycin B1; D000255:Adenosine Triphosphate; D019204:GTP-Binding Proteins; D006180:Proton-Translocating ATPases