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

Mahdokht Kohansal-Nodehi,Dominika Czernik,Henning Urlaub,Reinhard Jahn,John Je Chua

doi:10.7554/elife.14530

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.

Highlights

In chemical synapses, neurotransmitters are stored in synaptic vesicles and released by Ca2+-dependent exocytosis upon stimulation
Synaptic vesicles attach to specialized sites at the presynaptic plasma membrane, which is associated with activation of the exocytotic apparatus
Addition of KCl led to a fast increase in glutamate release that persisted for several minutes after depolarization and that was reduced in the absence of Ca2+ (Figure 1A)

Summary

Introduction

Neurotransmitters are stored in synaptic vesicles and released by Ca2+-dependent exocytosis upon stimulation. The components of the synaptic vesicle membrane are retrieved, mainly by clathrin-dependent endocytosis, and used to regenerate synaptic vesicles for another round of exo-endocytotic recycling (Sudhof, 2013; Rizzoli, 2014). The steps of the synaptic vesicle cycle have been studied in great detail. Synaptic vesicles attach to specialized sites at the presynaptic plasma membrane (active zones), which is associated with activation (priming) of the exocytotic apparatus. Functionally distinct pools of synaptic vesicles coexist in each nerve terminal, with only a small proportion of them participating in recycling under normal physiological conditions (Denker et al, 2011).

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Conclusion