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Intravesicular acidification correlates with binding of ADP-ribosylation factor to microsomal membranes
S Zeuzem1, P Feick, P Zimmermann
1Max-Planck-Institut für Biophysik, Frankfurt, Federal Republic of Germany.
Abstract:
The ADP-ribosylation factor (ARF), a highly conserved low molecular weight GTP-binding protein, has been implicated to function in intracellular protein transport to and within the Golgi complex. In pancreatic acinar cells the ARF is confined to the cytoplasmic faces of trans-Golgi stack membranes, a compartment known to maintain a low intravesicular pH, which is established by a chloride-dependent MgATP-driven proton pump. The present study shows that MgATP (2mM), but neither adenosine 5'-[gamma-thio]triphosphate in the presence of Mg2+ nor ATP in the absence of Mg2+, increases transfer of ARF from the surrounding medium into the vesicle membranes. The specific vacuolar-type proton pump inhibitor bafilomycin B1 (10 nM), the protonophore carbonylcyanide m-chlorophenylhydrazone (10 microM), and replacement of chloride in the incubation buffer by acetate or nitrate resulted in an almost complete inhibition of the MgATP-dependent association of ARF to the vesicle membranes. The results demonstrate that redistribution of ARF to the vesicle membrane correlates with the intravesicular pH established by a vacuolar-type H(+)-ATPase. The intravesicular pH appears to be one mechanism by which certain low molecular weight GTP-binding proteins become relocated from the cytosol to their specific membrane vesicles.
Insights
ADP-ribosylation factor (ARF) protein transfer to Golgi membranes is regulated by intravesicular pH. This pH is established by a proton pump, and its manipulation affects ARF protein localization.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- ADP-ribosylation factor (ARF) is a GTP-binding protein crucial for intracellular protein transport within the Golgi complex.
- In pancreatic acinar cells, ARF localizes to the trans-Golgi network, a compartment with a low intravesicular pH maintained by a proton pump.
- This low pH is established via a chloride-dependent MgATP-driven proton pump.
Purpose of the Study:
- To investigate the role of intravesicular pH in the membrane association of ARF.
- To determine if MgATP and proton pump activity influence ARF redistribution to vesicle membranes.
Main Methods:
- Assessing ARF transfer to vesicle membranes under various conditions, including MgATP presence/absence and with specific inhibitors.
- Utilizing MgATP, adenosine 5'-[gamma-thio]triphosphate, and bafilomycin B1 (proton pump inhibitor).
- Employing protonophores and altering buffer composition (chloride replacement) to modulate intravesicular pH.
Main Results:
- MgATP significantly increased ARF transfer to vesicle membranes, an effect dependent on Mg2+.
- Inhibition of the proton pump (bafilomycin B1), disruption of proton gradient (protonophore), and chloride replacement abolished MgATP-dependent ARF association.
- ARF redistribution to vesicle membranes directly correlated with the intravesicular pH maintained by the vacuolar-type H(+)-ATPase.
Conclusions:
- Intravesicular pH, regulated by the vacuolar H(+)-ATPase, is a key factor controlling ARF redistribution to Golgi vesicle membranes.
- Changes in intravesicular pH serve as a mechanism for relocating low molecular weight GTP-binding proteins like ARF from the cytosol to specific membrane compartments.
- This finding provides insight into the regulation of intracellular protein transport and protein-membrane interactions.