A Fine Balance of Synaptophysin Levels Underlies Efficient Retrieval of Synaptobrevin II to Synaptic Vesicles

Sarah L Gordon,Callista B Harper,Michael A Cousin,Karen J Smillie

doi:10.1371/journal.pone.0149457

Abstract

Synaptobrevin II (sybII) is a vesicular soluble NSF attachment protein receptor (SNARE) protein that is essential for neurotransmitter release, and thus its correct trafficking to synaptic vesicles (SVs) is critical to render them fusion competent. The SV protein synaptophysin binds to sybII and facilitates its retrieval to SVs during endocytosis. Synaptophysin and sybII are the two most abundant proteins on SVs, being present in a 1:2 ratio. Synaptophysin and sybII are proposed to form a large multimeric complex, and the copy number of the proteins in this complex is also in a 1:2 ratio. We investigated the importance of this ratio between these proteins for the localisation and trafficking of sybII in central neurons. SybII was overexpressed in mouse hippocampal neurons at either 1.6 or 2.15–2.35-fold over endogenous protein levels, in the absence or presence of varying levels of synaptophysin. In the absence of exogenous synaptophysin, exogenous sybII was dispersed along the axon, trapped on the plasma membrane and retrieved slowly during endocytosis. Co-expression of exogenous synaptophysin rescued all of these defects. Importantly, the expression of synaptophysin at nerve terminals in a 1:2 ratio with sybII was sufficient to fully rescue normal sybII trafficking. These results demonstrate that the balance between synaptophysin and sybII levels is critical for the correct targeting of sybII to SVs and suggests that small alterations in synaptophysin levels might affect the localisation of sybII and subsequent presynaptic performance.

Highlights

Neuronal activity relies on the efficient reformation of fusion-competent synaptic vesicles (SVs) to sustain neurotransmitter release
Synaptophysin expression levels are critical for retention of Synaptobrevin II (sybII) at nerve terminals Synaptophysin and sybII exist in a 1:2 ratio on SVs, which is reflected in the relative copy number of the two proteins when forming large multimeric complexes
This suggests that the relative proportion of each cargo molecule is key to normal neuronal function and that perturbation of this ratio may result in disrupted sybII trafficking

Summary

Introduction

Neuronal activity relies on the efficient reformation of fusion-competent synaptic vesicles (SVs) to sustain neurotransmitter release. This requires the packaging of vesicular membrane with the correct complement of SV cargo proteins at a defined stoichiometry [1,2]. It is becoming increasingly evident that protein-loading of SVs during endocytosis is a highly regulated process, requiring classical clathrin adaptor proteins, monomeric adaptor proteins, and specialised intrinsic vesicle proteins that traffic essential vesicular cargo [3,4]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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