Reconstituted synaptotagmin I mediates vesicle docking, priming, and fusion

Wang, Zhao; Liu, Huisheng; Gu, Yiwen; Chapman, Edwin R.

doi:10.1083/jcb.201104079

Cited by 94 publications

(101 citation statements)

References 58 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Consistent with the washer model, it is known that the initial docking of the synaptic vesicles to the plasma membrane is independent of SNARE complex assembly (18,51,52) and involves SYT from the synaptic vesicle binding the acidic lipids, including PIP 2 in the plasma membrane (9,(53)(54)(55)(56). Furthermore, although deletion of CPX markedly increases the amount of spontaneous transmitter release and reduces the amount of calcium-coupled release, much release still remains (provided SYT remains) (46,49,(57)(58)(59).…”

Section: Discussionmentioning

confidence: 55%

Calcium sensitive ring-like oligomers formed by synaptotagmin

Bello¹,

Auclair²,

Wang³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

159

View full text Add to dashboard Cite

The synaptic vesicle protein synaptotagmin-1 (SYT) is required to couple calcium influx to the membrane fusion machinery. However, the structural mechanism underlying this process is unclear.Here we report an unexpected circular arrangement (ring) of SYT's cytosolic domain (C2AB) formed on lipid monolayers in the absence of free calcium ions as revealed by electron microscopy. Rings vary in diameter from 18-43 nm, corresponding to 11-26 molecules of SYT. Continuous stacking of the SYT rings occasionally converts both lipid monolayers and bilayers into protein-coated tubes. Helical reconstruction of the SYT tubes shows that one of the C2 domains (most likely C2B, based on its biochemical properties) interacts with the membrane and is involved in ring formation, and the other C2 domain points radially outward. SYT rings are disrupted rapidly by physiological concentrations of free calcium but not by magnesium. Assuming that calcium-free SYT rings are physiologically relevant, these results suggest a simple and novel mechanism by which SYT regulates neurotransmitter release: The ring acts as a spacer to prevent the completion of the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) complex assembly, thereby clamping fusion in the absence of calcium. When the ring disassembles in the presence of calcium, fusion proceeds unimpeded. S ynaptotagmin-1 (SYT) is the calcium (Ca 2+ ) sensor that triggers synchronous release of neurotransmitters for synaptic transmission (1-4). It is a transmembrane protein, localized to the synaptic vesicles (1, 5), with tandem cytosolic C2 domains (C2A and C2B) that bind phospholipids in both a Ca 2+ -independent and a Ca 2+ -dependent manner (1, 6). The membrane-distal C2B domain interacts with acidic lipids such as phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to mediate efficient docking of the synaptic vesicles (7-10) before the influx of Ca 2+ ions. Recent studies (7,8,11,12) have located this calciumindependent membrane-binding site to a polybasic patch on the C2B domain (site I in Fig. 1A). The binding of Ca 2+ to the calcium-coordination pocket of the C2B domain (site II in Fig. 1A) triggers the insertion of flanking portions of site II into the plasma membrane, and this Ca 2+ -triggered membrane insertion is absolutely required for neurotransmitter release (13-16).The C2B domain also mediates the Ca 2+ -independent binding of SYT to neuronal soluble N-ethylmaleimide-sensitive factor activating protein receptor on the plasma membrane (t-SNARE) [syntaxin/ synaptosomal-associated protein 25 (SNAP25)], most likely via its interaction with SNAP25 (17-21). This interaction is believed to position SYT on the prefusion SNARE complexes to trigger rapid exocytosis in response to Ca 2+ (20,22). How the insertion of site II into the membrane bilayer is coupled to the completion of the SNARE assembly to release neurotransmitter is still unclear, although some key points have been established. In the prefusion state, the SNARE complexes...

show abstract

Section: Discussionmentioning

confidence: 55%

Calcium sensitive ring-like oligomers formed by synaptotagmin

Bello¹,

Auclair²,

Wang³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

159

View full text Add to dashboard Cite

show abstract

“…A Ca 2+ -independent role attributed to synaptotagmin was illustrated by SytI mutant, which lacks Ca 2+ binding in both its C2 domains and facilitates vesicle docking. 52 Ca 2+ -independent role of SytI is also supported by its ability to facilitate close membrane apposition in vitro even in the absence of calcium binding [24][25][26] and the recent identification of Ca 2+ -independent SNARE-binding motifs. 53 Syt XI may similarly facilitate lysosome docking to the cell membrane.…”

Section: Discussionmentioning

confidence: 99%

Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis

et al. 2015

View full text Add to dashboard Cite

Astrocytes are known to facilitate repair following brain injury; however, little is known about how injured astrocytes repair themselves. Repair of cell membrane injury requires Ca 2+ -triggered vesicle exocytosis. In astrocytes, lysosomes are the main Ca 2+ -regulated exocytic vesicles. Here we show that astrocyte cell membrane injury results in a large and rapid calcium increase. This triggers robust lysosome exocytosis where the fusing lysosomes release all luminal contents and merge fully with the plasma membrane. In contrast to this, receptor stimulation produces a small sustained calcium increase, which is associated with partial release of the lysosomal luminal content, and the lysosome membrane does not merge into the plasma membrane. In most cells, lysosomes express the synaptotagmin (Syt) isoform Syt VII; however, this isoform is not present on astrocyte lysosomes and exogenous expression of Syt VII on lysosome inhibits their exocytosis. Deletion of one of the most abundant Syt isoform in astrocyte -Syt XI -suppresses astrocyte lysosome exocytosis. This identifies lysosome as Syt XI-regulated exocytic vesicle in astrocytes. Further, inhibition of lysosome exocytosis (by Syt XI depletion or Syt VII expression) prevents repair of injured astrocytes. These results identify the lysosomes and Syt XI as the sub-cellular and molecular regulators, respectively of astrocyte cell membrane repair.

show abstract

“…In the absence of PI(4,5)P 2 , a large functional difference between Syt1-wt and Syt1-RQ was observed. Whereas Syt1-wt showed a minor impairment in docking efficiency as observed before for synthetic vesicle-vesicle tethering (Vennekate et al, 2012, Parisotto et al, 2012, Syt1-RQ was completely defective in docking SUVs to GUVs lacking PI(4,5)P 2 (Fig. 3C).…”

Section: Syt1-c2b Bottom Region Is Dispensable For Secretory Vesicle mentioning

confidence: 91%

A Post-Docking Role of Synaptotagmin 1-C2B Domain Bottom Residues R398/399 in Mouse Chromaffin Cells

Kedar

Munch

Weering³

et al. 2015

J. Neurosci.

View full text Add to dashboard Cite

Synaptotagmin-1 (Syt1) is the principal Ca 2ϩ sensor for vesicle fusion and is also essential for vesicle docking in chromaffin cells. Docking depends on interactions of the Syt1-C2B domain with the t-SNARE SNAP25/Syntaxin1 complex and/or plasma membrane phospholipids. Here, we investigated the role of the positively charged "bottom" region of the C2B domain, proposed to help crosslink membranes, in vesicle docking and secretion in mouse chromaffin cells and in cell-free assays. We expressed a double mutation shown previously to interfere with lipid mixing between proteoliposomes and with synaptic transmission, Syt1-R398/399Q (RQ), in syt1 null mutant cells. Ultrastructural morphometry revealed that Syt1-RQ fully restored the docking defect observed previously in syt1 null mutant cells, similar to wild type Syt1 (Syt1-wt). Small unilamellar lipid vesicles (SUVs) that contained the v-SNARE Synaptobrevin2 and Syt1-R398/399Q also docked to t-SNARE-containing giant vesicles (GUVs), similar to Syt1-wt. However, unlike Syt1-wt, Syt1-RQinduced docking was strictly PI(4,5)P 2 -dependent. Unlike docking, neither synchronized secretion in chromaffin cells nor Ca 2ϩ -triggered SUV-GUV fusion was restored by the Syt1 mutants. Finally, overexpressing the RQ-mutant in wild type cells produced no effect on either docking or secretion. We conclude that the positively charged bottom region in the C2B domain-and, by inference, Syt1-mediated membrane crosslinking-is required for triggering fusion, but not for docking. Secretory vesicles dock by multiple, PI(4,5)P 2 -dependent and PI(4,5)P 2 -independent mechanisms. The R398/399 mutations selectively disrupt the latter and hereby help to discriminate protein regions involved in different aspects of Syt1 function in docking and fusion.

show abstract

Reconstituted synaptotagmin I mediates vesicle docking, priming, and fusion

Abstract: In vitro reconstitution fusion assays incorporating full-length membrane-anchored synaptotagmin I clarify its role in several steps in the secretory pathway.

Cited by 94 publications

References 58 publications

Calcium sensitive ring-like oligomers formed by synaptotagmin

Calcium sensitive ring-like oligomers formed by synaptotagmin

Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis

A Post-Docking Role of Synaptotagmin 1-C2B Domain Bottom Residues R398/399 in Mouse Chromaffin Cells

Contact Info

Product

Resources

About