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Synaptic vesicles isolated from 32P-prelabeled synaptosomes contain a phosphoprotein of apparent M(r) 65,000 (pp65),
P Gómez-Puertas1, J Satrústegui, E Bogónez
1Departamento de Biología Molecular, Universidad Autonoma de Madrid, Spain.
Abstract:
In the present study, we have investigated the subcellular localization of pp65, a synaptosomal phosphoprotein of apparent M(r) 65,000. The results obtained strongly support that pp65 is localized to synaptic vesicles. The solubility properties of pp65, especially its partitioning into the detergent phase of Triton X-114, indicated that it is tightly associated with the membrane of synaptic vesicles. pp65 is multiply phosphorylated exclusively on serine. By studying the decay of labeled phosphate following incubation of 32P-prelabeled synaptosomes in the presence of cold inorganic phosphate, we have found that pp65 shows an unusually high turnover of phosphate. Exposure of synaptosomes to 1 microM phorbol 12-myristate 13-acetate prior to prelabeling with 32P(i) led to a reduction in the steady state phosphorylation of pp65, and tryptic/chymotryptic mapping was shown to selectively affect phosphopeptide 4. Identical results were obtained following incubation of synaptosomes with the protein kinase C (PKC) inhibitor, GF 109203 X. These results indicated that one of the protein kinases involved in steady state phosphorylation of pp65 is PKC-dependent or is PKC itself. Several characteristics of pp65 reported in the present study suggest a regulatory role in nerve terminal function.
Insights
Synaptic vesicle phosphoprotein pp65 is localized to synaptic vesicles and tightly associated with their membranes. Protein kinase C (PKC) influences its phosphorylation, suggesting a regulatory role in nerve terminal function.
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- Synaptosomes are crucial for neurotransmission.
- Synaptic vesicles are key components of nerve terminals.
- Phosphoproteins play vital roles in neuronal function.
Purpose of the Study:
- To determine the subcellular localization of the synaptosomal phosphoprotein pp65.
- To investigate the phosphorylation and regulation of pp65.
- To explore the potential role of pp65 in nerve terminal function.
Main Methods:
- Subcellular fractionation and Triton X-114 phase partitioning to assess pp65 localization.
- Radiolabeling with 32P(i) and phosphate turnover studies to analyze phosphorylation dynamics.
- Treatment with phorbol ester and protein kinase C inhibitor (GF 109203 X) followed by phosphopeptide mapping.
Main Results:
- pp65 is localized to synaptic vesicles and tightly associated with their membranes.
- pp65 exhibits high phosphate turnover, exclusively on serine residues.
- Phorbol ester and a PKC inhibitor reduced pp65 phosphorylation, implicating protein kinase C.
Conclusions:
- pp65 is a synaptic vesicle-associated phosphoprotein.
- Protein kinase C is involved in the regulation of pp65 phosphorylation.
- pp65 likely plays a regulatory role in nerve terminal function.