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Free keywords:
ras-related GTP-binding proteins; ADP-ribosylation factor; proton pump; bafilomycin; GTP-binding blots
Abstract:
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.
