Mechanism of membrane fusion::SNARE (protein)

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Mechanism of membrane fusion

Assembly

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Depiction of the formation of a trans-SNARE complex. Shows how Munc18 interacts with the SNARE proteins during complex formation.

SNARE proteins must assemble into trans-SNARE complexes so that they can provide the force that is necessary for vesicle fusion. The four α-helix domains (1 each from synaptobrevin and syntaxin, and 2 from SNAP-25) come together to form a coiled-coil motif. The rate-limiting step in the assembly process is the association of the syntaxin SNARE domain, since it is usually found in a "closed" state where it is incapable of interacting with other SNARE proteins.<ref name="Burkhardt_2008">{{#invoke:Citation/CS1|citation |CitationClass=journal }}</ref> When syntaxin is in an open state, trans-SNARE complex formation begins with the association of the four SNARE domains at their N-termini. The SNARE domains proceed in forming a coiled-coil motif in the direction of the C-termini of their respective domains.

The SM protein Munc18 is thought to play a role in assembly of the SNARE complex, although the exact mechanism by which it acts is still under debate. It is known that the clasp of Munc18 locks syntaxin in a closed conformation by binding to its α-helical SNARE domains, which inhibits syntaxin from entering SNARE complexes (thereby inhibiting fusion).<ref name="Burkhardt_2008"/> The clasp is also capable, however, of binding the entire four-helix bundle of the trans-SNARE complex. One hypothesis suggests that, during SNARE-complex assembly, the Munc18 clasp releases closed syntaxin, remains associated with the N-terminal peptide of syntaxin (allowing association of the syntaxin SNARE domain with other SNARE proteins), and then reattaches to the newly formed four-helix SNARE complex.<ref name="Südhof_2009">{{#invoke:Citation/CS1|citation |CitationClass=journal }}</ref> This possible mechanism of dissociation and subsequent re-association with the SNARE domains could be calcium-dependent.<ref name="Jahn_2012">{{#invoke:Citation/CS1|citation |CitationClass=journal }}</ref> This supports the idea that Munc18 plays a key regulatory role in vesicle fusion; under normal conditions the SNARE complex will be prevented from forming by Munc18, but when triggered the Munc18 will actually assist in SNARE-complex assembly and thereby act as a fusion catalyst.<ref name="Südhof_2009"/>

Zippering and fusion pore opening

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This figure provides a simple overview of the interaction of SNARE proteins with vesicles during exocytosis. Shows SNARE complex assembly, zippering, and disassembly.

Membrane fusion is an energetically demanding series of events, which requires translocation of proteins in the membrane and disruption of the lipid bilayer, followed by reformation of a highly curved membrane structure. The process of bringing together two membranes requires input energy to overcome the repulsive electrostatic forces between the membranes. The mechanism that regulates the movement of membrane associated proteins away from the membrane contact zone prior to fusion is unknown, but the local increase in membrane curvature is thought to contribute in the process. SNAREs generate energy through protein-lipid and protein-protein interactions which act as a driving force for membrane fusion.

One model hypothesizes that the force required to bring two membranes together during fusion comes from the conformational change in trans-SNARE complexes to form cis-SNARE complexes. The current hypothesis that describes this process is referred to as SNARE "zippering."<ref name="Chen_2001">{{#invoke:Citation/CS1|citation |CitationClass=journal }}</ref>

When the trans-SNARE complex is formed, the SNARE proteins are still found on opposing membranes. As the SNARE domains continue coiling in a spontaneous process, they form a much tighter, more stable four-helix bundle. During this "zippering" of the SNARE complex, a fraction of the released energy from binding is thought to be stored as molecular bending stress in the individual SNARE motifs. This mechanical stress is postulated to be stored in the semi-rigid linker regions between the transmembrane domains and the SNARE helical bundle.<ref name="pmid11349128">{{#invoke:Citation/CS1|citation |CitationClass=journal }}</ref><ref>{{#invoke:Citation/CS1|citation |CitationClass=journal }}</ref> The energetically unfavorable bending is minimized when the complex moves peripherally to the site of membrane fusion. As a result, relief of the stress overcomes the repulsive forces between the vesicle and the cell membrane and presses the two membranes together.<ref>{{#invoke:Citation/CS1|citation |CitationClass=journal }}</ref>

Several models to explain the subsequent step - the formation of stalk and fusion pore - have been proposed. However, the exact nature of these processes remains debated. In accordance with the "zipper" hypothesis, as the SNARE complex forms, the tightening helix bundle puts torsional force on the transmembrane (TM) domains domains of synaptobrevin and syntaxin.<ref name="pmid24985331">{{#invoke:Citation/CS1|citation |CitationClass=journal }}</ref> This causes the TM domains to tilt within the separate membranes as the proteins coil more tightly. The unstable configuration of the TM domains eventually causes the two membranes to fuse and the SNARE proteins come together within the same membrane, which is referred to as a "cis"-SNARE complex.<ref>{{#invoke:citation/CS1|citation |CitationClass=book }}</ref> As a result of the lipid rearrangement, a fusion pore opens and allows the chemical contents of the vesicle to leak into the outside environment.

The continuum explanation of stalk formation suggests that membrane fusion begins with an infinitesimal radius until it radially expands into a stalk-like structure. However, such a description fails to take into account the molecular dynamics of membrane lipids. Recent molecular simulations show that the close proximity of the membranes allows the lipids to splay, where a population of lipids insert one their hydrophobic tails into the neighboring membrane - effectively keeping a "foot" in each membrane. The resolution of the splayed lipid state proceeds spontaneously to form the stalk structure. In this molecular view, the splayed-lipid intermediate state is the rate determining barrier rather than the formation of the stalk, which now becomes the free energy minimum. The energetic barrier for establishment of the splayed-lipid conformation is directly proportional to the intermembrane distance. The SNARE complexes and their pressing of the two membranes together, therefore, could provide the free energy required to overcome the barrier.<ref>{{#invoke:Citation/CS1|citation |CitationClass=journal }}</ref>

Disassembly

The energy input that is required for SNARE-mediated fusion to take place comes from SNARE-complex disassembly. The suspected energy source is N-ethylmaleimide-sensitive factor (NSF), an ATPase that is involved with membrane fusion. NSF homohexamers, along with the NSF cofactor α-SNAP, bind and dissociate the SNARE complex by coupling the process with ATP hydrolysis.<ref name="Söllner_1993">{{#invoke:Citation/CS1|citation |CitationClass=journal }}</ref> This process allows for reuptake of synaptobrevin for further use in vesicles, whereas the other SNARE proteins remain associated with the cell membrane.

The dissociated SNARE proteins have a higher energy state than the more stable cis-SNARE complex. It is believed that the energy that drives fusion is derived from the transition to a lower energy cis-SNARE complex. The ATP hydrolysis-coupled dissociation of SNARE complexes is an energy investment that can be compared to "cocking the gun" so that, once vesicle fusion is triggered, the process takes place spontaneously and at optimum velocity. A comparable process takes place in muscles, in which the myosin heads must first hydrolyze ATP in order to adapt the necessary conformation for interaction with actin and the subsequent power stroke to occur.


SNARE (protein) sections
Intro   Types    Structure    Membrane fusion    Components    Mechanism of membrane fusion   Regulatory Effects on Exocytosis   Toxins    Role in neurotransmitter release    Role in autophagy   References  External links  

Mechanism of membrane fusion
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