Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis

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Abstract

Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity.

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Mahdokht Kohansal-Nodehi

Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

Competing interests
No competing interests declared.
John JE Chua

Interactomics and Intracellular Trafficking laboratory, Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

Competing interests
No competing interests declared.
Henning Urlaub

Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

Competing interests
No competing interests declared.
Reinhard Jahn

Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

Competing interests
Reinhard Jahn, Reviewing editor, eLife.
Dominika Czernik

Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany

For correspondence
dominika.czernik@mpibpc.mpg.de

Competing interests
No competing interests declared.

Ethics

Animal experimentation: All animal procedures used here fully comply with the guidelines as stipulated in the section 4 of the Animal Welfare Law of the Federal Republic of Germany (section 4 of TierSchG, Tierschutzgesetz der Bundesrepublik Deutschland). All procedures were performed in the animal facility at the Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany registered accordingly to the section 11 Abs. 1 TierSchG as documented by 39 20 00_2a Si/rö, dated 11th Dec 2013 ("Erlaubnis, Wirbeltiere zur Versuchszwecken zu züchten und zu halten"), by the Veterinär- und Verbraucherschutzamt für den Landkreis und die Stadt Göttingen and examined regularly by the supervisory veterinary authority of the Landkreis Göttingen. All procedures were supervised by the animal welfare officer and the animal welfare committee of the Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany established accordingly to the TierSchG and the regulation about animal used in experiments, dated on 1st Aug 2013 (TierSchVersV, Tierschutz-Versuchstier-Verordung).

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