Abstract
Synaptotagmin 1 (Syt1), a major Ca2+ sensor in neuroexocytosis, utilizes SNARE- and membrane-binding to regulate vesicle fusion, a required process for neurotransmitter release at the synapse. However, the mechanism by which Syt1 orchestrates SNARE- and membrane- binding to control individual vesicle fusion steps is still unclear. In this study, we used a number of single vesicle assays that can differentiate intermediates of neuroexocytosis, to focus on Syt1 mutants that might impair Syt1-SNARE/PIP2 interaction, Ca2+-binding, or membrane penetration. Our results show that, although putative Syt1-SNARE/PIP2 coupling through the polybasic region of the C2B domain is critical for vesicle docking, its disruption does not affect content release. In contrast, Ca2+-binding and membrane-penetration mutants significantly reduce content release. Our results thus delineate multiple functions of Syt1 along the pathway of Ca2+-triggered exocytosis in unprecedented detail.
Highlights
Synaptotagmin 1 (Syt1), a major Ca21 sensor in neuroexocytosis, utilizes SNARE- and membrane-binding to regulate vesicle fusion, a required process for neurotransmitter release at the synapse
Putative Syt1-SNARE/PIP2 coupling through the polybasic region of the C2B domain is critical for vesicle docking, its disruption does not affect content release
In this work, our single vesicle fusion assay revealed that mutations in the polybasic region in Syt[1] cause reduced SNARE and PIP2 binding and result in an apparent decrease in vesicle docking, but produce little change in the content release
Summary
Synaptotagmin 1 (Syt1), a major Ca21 sensor in neuroexocytosis, utilizes SNARE- and membrane-binding to regulate vesicle fusion, a required process for neurotransmitter release at the synapse. Syt[1] could bind to the negatively charged lipid PIP2 (Phosphatidylinositol 4,5-bisphosphate) on the plasma membrane probably via the same polybasic region on the C2B domain in the absence of Ca21, while the loop regions could penetrate into the acidic membrane upon Ca21 binding[20,21,22,23,24] In functional studies, both SNARE and membrane binding by the C2 domains have proven essential in achieving vesicle fusion[18,25,26,27,28,29,30]. Because of experimental difficulties for previous ensemble fusion assays in resolving individual steps along the fusion pathway, many ambiguities still remain with respect to understanding the molecular origins of the Syt1’s involvement in individual steps of Ca21-triggered neuroexocytosis
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