Ca2+ Regulates the Drosophila Stoned-A and Stoned-B Proteins Interaction with the C2B Domain of Synaptotagmin-1

Carolina Soekmadji,Clement Angkawidjaja,Leonard E Kelly,Karl-Wilhelm Koch

doi:10.1371/journal.pone.0038822

Carolina Soekmadji, Clement Angkawidjaja + Show 2 more

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https://doi.org/10.1371/journal.pone.0038822

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Journal: PloS one	Publication Date: Jun 12, 2012
Citations: 27	License type: CC BY 4.0

Affiliation: University of Melbourne, Osaka University

Abstract

The dicistronic Drosophila stoned gene is involved in exocytosis and/or endocytosis of synaptic vesicles. Mutations in either stonedA or stonedB cause a severe disruption of neurotransmission in fruit flies. Previous studies have shown that the coiled-coil domain of the Stoned-A and the µ-homology domain of the Stoned-B protein can interact with the C2B domain of Synaptotagmin-1. However, very little is known about the mechanism of interaction between the Stoned proteins and the C2B domain of Synaptotagmin-1. Here we report that these interactions are increased in the presence of Ca2+. The Ca2+-dependent interaction between the µ-homology domain of Stoned-B and C2B domain of Synaptotagmin-1 is affected by phospholipids. The C-terminal region of the C2B domain, including the tryptophan-containing motif, and the Ca2+ binding loop region that modulate the Ca2+-dependent oligomerization, regulates the binding of the Stoned-A and Stoned-B proteins to the C2B domain. Stoned-B, but not Stoned-A, interacts with the Ca2+-binding loop region of C2B domain. The results indicate that Ca2+-induced self-association of the C2B domain regulates the binding of both Stoned-A and Stoned-B proteins to Synaptotagmin-1. The Stoned proteins may regulate sustainable neurotransmission in vivo by binding to Ca2+-bound Synaptotagmin-1 associated synaptic vesicles.

Highlights

Synaptotagmin-1 (SYT-1) is primarily associated with Ca2+dependent exocytosis of synaptic vesicles (SV)
We have found that Ca2+ greatly increases the binding of STNA and STNB to the C2B domain of SYT-1, that these interactions are primarily driven by a Ca2+-dependent self-association of the C2B domains of SYT-1 molecules and that the likely binding site for STNB resides within the Ca2+-binding loops of the dimeric or multimeric C2B protein domains
Phospholipids abolish the binding of STNB to C2B SYT-1 A recent study reported that m2 cannot replace the function of the mHD of STNB in vivo [34], and that the binding conditions of m2 of AP-2 to the C2B domain require Ca2+ and phosphatidyl serine (PS) [20]

Summary

Introduction

Synaptotagmin-1 (SYT-1) is primarily associated with Ca2+dependent exocytosis of synaptic vesicles (SV). An integral membrane component of SV, SYT-1 is essential for fast Ca2+dependent release of neurotransmitter in Drosophila, C. elegans, and mouse [1,2,3,4,5,6,7] Structurally synaptotagmins consist of a short luminal domain, a single transmembrane segment, and two tandem C2 domains (C2A and C2B). These C2 domains form Ca2+-dependent complexes with the SNARE proteins and trigger exocytosis [8,9,10]. Further study has shown the interaction of AP2 with SYT-1 to be Ca2+ and phosphatidylserine-dependent and to involve the m2 subunit of AP-2 interacting with the lysine-rich motif of C2B SYT-1 [20]

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