Membrane Fusion Intermediates via Directional and Full Assembly of the SNARE Complex

Hernandez, Javier M.; Stein, Alexander; Behrmann, Elmar; Riedel, Dietmar; Cypionka, Anna; Farsi, Zohreh; Walla, Peter Jomo; Raunser, Stefan; Jahn, Reinhard

doi:10.1126/science.1221976

Cited by 222 publications

(378 citation statements)

References 53 publications

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“…However, when combining SNAP23 and STX3 vesicles with vesicles that contain a phosphomimetic version of VAMP8, VAMP8Glu (T47E, T53E, S54E), both the kinetics of content‐mixing (Fig 3B) and lipid‐mixing (Fig EV5B) were reduced, indicating that the kinetics of fusion is reduced by phosphorylation of these VAMP8 sites. Surprisingly, the effect of the phosphomimetic mutations is not as severe as deletion mutants in the C‐terminal end of the neuronal SNARE complex that have been studied previously (Hernandez et al , 2012). In accordance with the reduced fusion kinetics, SDS‐resistant SNARE complexes were formed when combining SNAP23, STX3, and VAMP8 (Fig EV5C) and VAMP8Glu was less efficient in forming SNARE complexes when compared to VAMP8 and VAMP8Ala (T47A, T53A, S54A).…”

Section: Resultsmentioning

confidence: 80%

“…Incomplete zippering of the SNARE complex toward the C‐terminal end may result in docked vesicles that fail to fuse with the plasma membrane (Hernandez et al , 2012). To test the fusogenic activity of VAMP8, we used in vitro ensemble content‐ and lipid‐mixing assays with SNARE proteins reconstituted in proteoliposomes (Materials and Methods) (Diao et al , 2015).…”

Section: Resultsmentioning

confidence: 99%

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Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

et al. 2016

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Membrane fusion is essential for eukaryotic life, requiring SNARE proteins to zipper up in an α‐helical bundle to pull two membranes together. Here, we show that vesicle fusion can be suppressed by phosphorylation of core conserved residues inside the SNARE domain. We took a proteomics approach using a PKCB knockout mast cell model and found that the key mast cell secretory protein VAMP8 becomes phosphorylated by PKC at multiple residues in the SNARE domain. Our data suggest that VAMP8 phosphorylation reduces vesicle fusion in vitro and suppresses secretion in living cells, allowing vesicles to dock but preventing fusion with the plasma membrane. Markedly, we show that the phosphorylation motif is absent in all eukaryotic neuronal VAMPs, but present in all other VAMPs. Thus, phosphorylation of SNARE domains is a general mechanism to restrict how much cells secrete, opening the door for new therapeutic strategies for suppression of secretion.

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Section: Resultsmentioning

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

et al. 2016

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“…The directional assembly then proceeds toward the C termini, resulting in a pulling force between the membranes that leads to their fusion (4,5). There is some consensus that the highly exergonic nature of the assembly of the SNARE complex provides the energy for overcoming the barrier for fusion (6,7), although identification of putative fusion intermediates at molecular resolution as well as force measurement experiments suggest multiple energy barriers are present (7)(8)(9)(10).…”

Section: Exocytosis | Fusion Intermediate | Liposome Dockingmentioning

confidence: 99%

“…To overcome this, SNARE complex formation can be enhanced by using the ΔN complex, which accelerates this process by at least 30-fold (26). In a dilute liposome regime (0.02-0.1 mM total lipid) we have previously found that with the ΔN complex the rate of docking is accelerated to an extent similar to the rate of fusion, a kinetic condition known as partial-rate limiting (8,30).…”

Section: Significancementioning

confidence: 99%

“…We next asked whether membrane curvature stress has an impact on the effect of SNARE density on lipid mixing. We prepared large (>80-nm diameter) liposomes that have approximately twofold less curvature than the small liposomes used previously (8). To rigorously compare the effect of curvature on fusion, we first recorded fusion single events by TIRF microscopy with small and large syb liposomes as they fuse with a planar supported bilayer with reconstituted ΔN complexes.…”

Section: Large Liposomes Require More Snares For Maximal Lipid Mixingmentioning

confidence: 99%

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Variable cooperativity in SNARE-mediated membrane fusion

Hernandez

Kreutzberger

Kiessling

et al. 2014

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

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The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex drives the majority of intracellular and exocytic membrane fusion events. Whether and how SNAREs cooperate to mediate fusion has been a subject of intense study, with estimates ranging from a single SNARE complex to 15. Here we show that there is no universally conserved number of SNARE complexes involved as revealed by our observation that this varies greatly depending on membrane curvature. When docking rates of small (∼40 nm) and large (∼100 nm) liposomes reconstituted with different synaptobrevin (the SNARE present in synaptic vesicles) densities are taken into account, the lipid mixing efficiency was maximal with small liposomes with only one synaptobrevin, whereas 23-30 synaptobrevins were necessary for efficient lipid mixing in large liposomes. Our results can be rationalized in terms of strong and weak cooperative coupling of SNARE complex assembly where each mode implicates different intermediate states of fusion that have been recently identified by electron microscopy. We predict that even higher variability in cooperativity is present in different physiological scenarios of fusion, and we further hypothesize that plasticity of SNAREs to engage in different coupling modes is an important feature of the biologically ubiquitous SNARE-mediated fusion reactions.M embrane fusion is an essential reaction common to intracellular trafficking and exocytosis in eukaryotic cells. Although the process involves an intricate interplay of several proteins, the fusion of membranes is dependent on the conserved family of proteins known as soluble N-ethylmaleimide-sensitive factor attachment protein receptors, or SNAREs (1, 2). In the important case of the fusion of synaptic vesicles (SVs), the SNAREs responsible are vesicular synaptobrevin 2 (syb) and plasma membrane proteins SNAP-25A (SN25) and syntaxin-1A (syx). A critical intermediate seems to be an acceptor complex consisting of a threehelix bundle formed by a 1:1 syx:SN25 complex, which serves as a binding site for syb (3,4). According to the zipper hypothesis, the N termini of syb and the 1:1 syx:SN25 complex nucleate to form a parallel four-helix bundle called the SNARE complex. The directional assembly then proceeds toward the C termini, resulting in a pulling force between the membranes that leads to their fusion (4, 5). There is some consensus that the highly exergonic nature of the assembly of the SNARE complex provides the energy for overcoming the barrier for fusion (6, 7), although identification of putative fusion intermediates at molecular resolution as well as force measurement experiments suggest multiple energy barriers are present (7-10).The question of whether and how SNAREs cooperate to mediate fusion has received substantial attention. Although some studies have left open the possibility that the number of SNARE complexes that cooperate during fusion is variable (11, 12), much attention has been given to the notion of a preferred number of SNARE complexes, ...

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The fusion pore, 60 years after the first cartoon

Sharma

Lindau

2018

FEBS Letters

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Neurotransmitter release occurs in the form of quantal events by fusion of secretory vesicles with the plasma membrane, and begins with the formation of a fusion pore that has a conductance similar to that of a large ion channel or gap junction. In this review, we propose mechanisms of fusion pore formation and discuss their implications for fusion pore structure and function. Accumulating evidence indicates a direct role of soluble N-ethylmaleimide-sensitive-factor attachment receptor proteins in the opening of fusion pores. Fusion pores are likely neither protein channels nor purely lipid, but are of proteolipidic composition. Future perspectives to gain better insight into the molecular structure of fusion pores are discussed.

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Membrane Fusion Intermediates via Directional and Full Assembly of the SNARE Complex

Cited by 222 publications

References 53 publications

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

Variable cooperativity in SNARE-mediated membrane fusion

The fusion pore, 60 years after the first cartoon

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