Calcium-independent stimulation of membrane fusion and SNAREpin formation by synaptotagmin I

Lara K. Mahal; Sonia M. Sequeira; Jodi M. Gureasko; Thomas H. Söllner

doi:10.1083/jcb.200203135

Calcium-independent stimulation of membrane fusion and SNAREpin formation by synaptotagmin I

The Journal of Cell Biology ◽

10.1083/jcb.200203135 ◽

2002 ◽

Vol 158 (2) ◽

pp. 273-282 ◽

Cited By ~ 71

Author(s):

Lara K. Mahal ◽

Sonia M. Sequeira ◽

Jodi M. Gureasko ◽

Thomas H. Söllner

Keyword(s):

Membrane Fusion ◽

Synaptic Vesicle ◽

Fusion Reaction ◽

Snare Proteins ◽

Calcium Sensor ◽

Synaptic Vesicle Protein ◽

Synaptotagmin I ◽

In Trans ◽

Liposome Fusion ◽

Stimulation Of

Ñeurotransmitter release requires the direct coupling of the calcium sensor with the machinery for membrane fusion. SNARE proteins comprise the minimal fusion machinery, and synaptotagmin I, a synaptic vesicle protein, is the primary candidate for the main neuronal calcium sensor. To test the effect of synaptotagmin I on membrane fusion, we incorporated it into a SNARE-mediated liposome fusion assay. Synaptotagmin I dramatically stimulated membrane fusion by facilitating SNAREpin zippering. This stimulatory effect was topologically restricted to v-SNARE vesicles (containing VAMP 2) and only occurred in trans to t-SNARE vesicles (containing syntaxin 1A and SNAP-25). Interestingly, calcium did not affect the overall fusion reaction. These results indicate that synaptotagmin I can directly accelerate SNARE-mediated membrane fusion and raise the possibility that additional components might be required to ensure tight calcium coupling.

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Reconstituted synaptotagmin I mediates vesicle docking, priming, and fusion

The Journal of Cell Biology ◽

10.1083/jcb.201104079 ◽

2011 ◽

Vol 195 (7) ◽

pp. 1159-1170 ◽

Cited By ~ 72

Author(s):

Zhao Wang ◽

Huisheng Liu ◽

Yiwen Gu ◽

Edwin R. Chapman

Keyword(s):

Synaptic Transmission ◽

Membrane Fusion ◽

Synaptic Vesicle ◽

Cytoplasmic Domain ◽

Snare Proteins ◽

Antagonist Activity ◽

Vesicle Docking ◽

Synaptotagmin I ◽

Target Membrane

The synaptic vesicle protein synaptotagmin I (syt) promotes exocytosis via its ability to penetrate membranes in response to binding Ca2+ and through direct interactions with SNARE proteins. However, studies using full-length (FL) membrane-embedded syt in reconstituted fusion assays have yielded conflicting results, including a lack of effect, or even inhibition of fusion, by Ca2+. In this paper, we show that reconstituted FL syt promoted rapid docking of vesicles (<1 min) followed by a priming step (3–9 min) that was required for subsequent Ca2+-triggered fusion between v- and t-SNARE liposomes. Moreover, fusion occurred only when phosphatidylinositol 4,5-bisphosphate was included in the target membrane. This system also recapitulates some of the effects of syt mutations that alter synaptic transmission in neurons. Finally, we demonstrate that the cytoplasmic domain of syt exhibited mixed agonist/antagonist activity during regulated membrane fusion in vitro and in cells. Together, these findings reveal further convergence of reconstituted and cell-based systems.

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The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores

10.1101/623827 ◽

2019 ◽

Author(s):

Zhenyong Wu ◽

Nadiv Dharan ◽

Sathish Thiyagarajan ◽

Ben O’Shaughnessy ◽

Erdem Karatekin

Keyword(s):

Membrane Fusion ◽

Snare Complex ◽

Snare Proteins ◽

Fusion Pore ◽

Calcium Sensor ◽

Fusion Reactions ◽

Synaptotagmin 1 ◽

Pore Opening ◽

Pore Dilation ◽

Fusion Pores

ABSTRACTAll membrane fusion reactions proceed through an initial fusion pore, including calcium-triggered vesicular release of neurotransmitters and hormones. Expansion of this small pore to release cargo molecules is energetically costly and regulated by cells, but the mechanisms are poorly understood. Here we show that the neuronal/exocytic calcium sensor Synaptotagmin-1 (Syt1) promotes expansion of fusion pores induced by SNARE proteins, beyond its established role in coupling calcium influx to fusion pore opening. Our results suggest that fusion pore dilation by Syt1 requires interactions with SNAREs, PI(4,5)P2, and calcium. Pore opening was abolished by a mutation of the tandem C2 domain (C2AB) hydrophobic loops of Syt1, suggesting that their calcium-induced insertion into the membrane is required for pore opening. We propose that loop insertion is also required for pore expansion, but through a distinct mechanism. Mathematical modelling suggests that membrane insertion re-orients the C2 domains bound to the SNARE complex, rotating the SNARE complex so as to exert force on the membranes in a mechanical lever action that increases the intermembrane distance. The increased membrane separation provokes pore dilation to offset a bending energy penalty. We conclude that Syt1 assumes a critical role in calcium-dependent fusion pore dilation during neurotransmitter and hormone release.SIGNIFICANCE STATEMENTMembrane fusion is a fundamental biological process, required for development, infection by enveloped viruses, fertilization, intracellular trafficking, and calcium-triggered release of neurotransmitters and hormones when cargo-laden vesicles fuse with the plasma membrane. All membrane fusion reactions proceed through an initial, nanometer-sized fusion pore which can flicker open-closed multiple times before expanding or resealing. Pore expansion is required for efficient cargo release, but underlying mechanisms are poorly understood. Using a combination of single-pore measurements and quantitative modeling, we suggest that a complex between the neuronal calcium sensor Synaptotagmin-1 and the SNARE proteins together act as a calcium-sensitive mechanical lever to force the membranes apart and enlarge the pore.

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Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1703240114 ◽

2017 ◽

Vol 114 (30) ◽

pp. 8023-8028 ◽

Cited By ~ 23

Author(s):

Nicole Hams ◽

Murugesh Padmanarayana ◽

Weihong Qiu ◽

Colin P. Johnson

Keyword(s):

Calcium Channel ◽

Membrane Fusion ◽

Single Molecule ◽

Calcium Influx ◽

Synaptic Proteins ◽

Snare Proteins ◽

Scaffolding Protein ◽

Calcium Sensor ◽

Significant Difference ◽

Titration Assay

Sensory hair cells rely on otoferlin as the calcium sensor for exocytosis and encoding of sound preferentially over the neuronal calcium sensor synaptotagmin. Although it is established that synaptotagmin cannot rescue the otoferlin KO phenotype, the large size and low solubility of otoferlin have prohibited direct biochemical comparisons that could establish functional differences between these two proteins. To address this challenge, we have developed a single-molecule colocalization binding titration assay (smCoBRA) that can quantitatively characterize full-length otoferlin from mammalian cell lysate. Using smCoBRA, we found that, although both otoferlin and synaptotagmin bind membrane fusion SNARE proteins, only otoferlin interacts with the L-type calcium channel Cav1.3, showing a significant difference between the synaptic proteins. Furthermore, otoferlin was found capable of interacting with multiple SNARE and Cav1.3 proteins simultaneously, forming a heterooligomer complex. We also found that a deafness-causing missense mutation in otoferlin attenuates binding between otoferlin and Cav1.3, suggesting that deficiencies in this interaction may form the basis for otoferlin-related hearing loss. Based on our results, we propose a model in which otoferlin acts as a calcium-sensitive scaffolding protein, localizing SNARE proteins proximal to the calcium channel so as to synchronize calcium influx with membrane fusion. Our findings also provide a molecular-level explanation for the observation that synaptotagmin and otoferlin are not functionally redundant. This study also validates a generally applicable methodology for quantitatively characterizing large, multivalent membrane proteins.

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The Tlg SNARE complex is required for TGN homotypic fusion

The Journal of Cell Biology ◽

10.1083/jcb.200104093 ◽

2001 ◽

Vol 155 (6) ◽

pp. 969-978 ◽

Cited By ~ 29

Author(s):

Jason H. Brickner ◽

Jennifer M. Blanchette ◽

György Sipos ◽

Robert S. Fuller

Keyword(s):

Membrane Fusion ◽

Fusion Reaction ◽

Snare Complex ◽

Snare Proteins ◽

Attachment Protein ◽

Trans Golgi Network ◽

Rab Protein ◽

Protein Receptor ◽

Golgi Network ◽

Antibody Inhibition

Using a new assay for membrane fusion between late Golgi/endosomal compartments, we have reconstituted a rapid, robust homotypic fusion reaction between membranes containing Kex2p and Ste13p, two enzymes resident in the yeast trans-Golgi network (TGN). Fusion was temperature, ATP, and cytosol dependent. It was inhibited by dilution, Ca+2 chelation, N-ethylmaleimide, and detergent. Coimmunoisolation confirmed that the reaction resulted in cointegration of the two enzymes into the same bilayer. Antibody inhibition experiments coupled with antigen competition indicated a requirement for soluble NSF attachment protein receptor (SNARE) proteins Tlg1p, Tlg2p, and Vti1p in this reaction. Membrane fusion also required the rab protein Vps21p. Vps21p was sufficient if present on either the Kex2p or Ste13p membranes alone, indicative of an inherent symmetry in the reaction. These results identify roles for a Tlg SNARE complex composed of Tlg1p, Tlg2p, Vti1p, and the rab Vps21p in this previously uncharacterized homotypic TGN fusion reaction.

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