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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

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Summary

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 MgATPγS nor ATP in the absence of Mg2+, increases association of 19- and 21-kDa smg-proteins to the vesicle membrane as monitored by their [α-32P]GTP binding. The affinity of smg-proteins for [α-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 NO −3 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.

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Authors and Affiliations

  1. Max-Planck-Institut für Biophysik, D-6000, Frankfurt am Main 70, Germany

    Stefan Zeuzem, Petra Zimmermann & Irene Schulz

Authors
  1. Stefan Zeuzem
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  2. Petra Zimmermann
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  3. Irene Schulz
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Additional information

The arf-antibodies were kindly supplied by Dr. R.A. Kahn. We thank Prof. Dr. D. Gallwitz, Dr. R. Jahn, and Dr. E.G. Lapetina for kindly providing the ypt 1-, rab 3-, and rap 1-antibodies, respectively. ADP-ribosyltransferase C3 from Clostridium botulinum was kindly supplied by Prof. Dr. K. Aktories. This work was supported by the Jung-Stiftung für Wissenschaft und Forschung. S.Z. was supported by a grant of the Deutsche Forschungsgemeinschaft (Ze 237/3-1).

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Zeuzem, S., Zimmermann, P. & Schulz, I. 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. J. Membarin Biol. 125, 231–241 (1992). https://doi.org/10.1007/BF00236436

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  • DOI: https://doi.org/10.1007/BF00236436

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