1 Introduction
Membranes – two-dimensional bilayers consisting of lipids, proteins, and other molecules – are one of the key building blocks in various different life forms and are impermeable for most larger molecules and ions. They separate the interior of cells from the surrounding media and form highly structured compartments inside the cell where different metabolic reactions can take place simultaneously. The controlled exchange of small molecules and ions with the cell exterior and their transport between the compartments is mediated by ion channels and carrier proteins embedded inside the membrane. In contrast to that, larger molecules are filled into vesicles that are released from the host membrane via a process called budding and deliver the cargo by fusing with the target membrane. These two processes need to be highly regulated in order to prevent the fusion of liposomes with the wrong compartment and thus interfering with its functionality.
1.1 SNARE-mediated membrane fusion
A special case – the fusion of vesicles with the plasma membrane – is also referred to as exocytosis and can be found at the presynaptic membrane of synaptic boutons. Here, neurotransmitter filled vesicles fuse with the target membrane to release their cargo into the synaptic cleft. However, at the resting state membranes are ~10-20 nm apart from each other, a distance that has to be overcome for fusion to occur. This renders the process highly endergonic, as the reduction to a distance of few nanometers is e.g. concomitant with the dehydration of the lipid head groups. This raises the question, what drives the fusion of synaptic vesicles and how is it controlled?
The way for identifying the key players of membrane fusion was cleared by Rothman and coworkers by isolating the N-ethylmaleimide-sensitive factor (NSF) due to its ability to restore vesicle transport after deactivation with N-ethylmaleimide (NEM) and identifying its role in the fusion process.[1,2] Further studies showed that NSF binds to the target membrane via the soluble NSF attachment protein (SNAP) and that functional transport requires SNAP activity.[3] This led to the discovery of the receptors for SNAP in synapses called soluble N-ethylmaleimide-sensitive factor attachment receptor proteins (SNAREs): syntaxin 1A (syx 1A), SNAP25 (synaptosomal associated protein of 25 kDa), and synaptobrevin 2 (syb 2).[4] These proteins turned out to catalyze the process of neurotransmitter release at the presynaptic membrane together with different regulatory proteins, such as muncs, complexin, and synaptotagmin
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(Figure 1.1).[5,6] Briefly, vesicles are docked and primed in the active zone, also called readily releasable pool, the arriving of an action potential from the axon then triggers the opening of Ca2+ channels at the presynaptic membrane, and the influx of Ca2+ in turn stimulates the release of neurotransmitters into the synaptic cleft.[7,8] While this process is highly regulated by different proteins, early studies revealed that only the aforementioned three SNARE proteins are needed to drive membrane fusion.[9,10]
Figure 1.1 Schematic illustration of the process of exocytosis at synapses. (a,b) Priming of SNAREs through Munc 13 and 18 close to the presynaptic membrane. (c,d) SNAREs do not undergo fusion yet but are held in place by presumably complexin and synaptotagmin, until the influx of Ca2+ leads to the fusion of the opposing membranes.
(d) Consequently, vesicles release the neurotransmitters into the synaptic cleft where they dock to receptors at the postsynaptic membrane. Adapted from Munson.[6]
But what is the underlying mechanisms by which the interaction of syb 2, syx 1A, and SNAP25 overcomes the energy barrier of fusion? Söllner et al. discovered a 1:1:1 stoichiometry of the three SNARE proteins, of which syb 2 is anchored inside the vesicular membrane via a transmembrane domain (TMD) and is therefore also called vesicular SNARE (v-SNARE).[4,8]
On the other site syx 1A and SNAP25 can be found at the presynaptic membrane (t-SNAREs), of which syx 1A also contains a TMD while SNAP25 is connected via a palmitoylated linker region to the target membrane.[8] This linker region connects two 60-70 aa long coiled coil structures named SNARE-motifs that can also be found in syb 2 and syx 1A, the latter of which additionally contains a regulatory Habc domain connected to the N-terminus.[11] These in total four SNARE motifs are highly conserved in the family of SNARE proteins and are, except for syx 1A, largely unstructured in solution (Figure 1.2 I) but form a very stable tetrameric complex with largely alphahelical content upon interaction (Figure 1.2 III).[5,12–15] This complex contains 16 layers of mainly hydrophobic residues facing inwards, except for one hydrophilic 0-layer
3 with three glutamine (Q) and one arginine (R) residue.[16–18] Based on this 0-layer SNAREs are also divided into the four classes Qa,b,c and R-SNARE. The transformation from largely unstructured to a tight 4 -helix bundle starts with the interaction of the N-termini of the SNARE domains and proceeds to the C-terminal end in a zippering kind of fashion (Figure 1.2 II).[19,20] This process pulls the opposing membranes into close proximity and thus the energy released during complex formation subsequently leads to membrane fusion.[13,21,22] During fusion the SNARE-complex changes its conformation from a high energy trans-configuration into a low energy cis-configuration where both TMDs are located inside the same lipid bilayer (Figure 1.2 II, III).[8] The SNARE-complex is afterwards disassembled by the AAA+-ATPase NSF together with its SNAP receptor, vesicles and syb 2 retrieved from the target membrane, and liposomes reloaded with neurotransmitters to be ready for another fusion cycle.[7,8]
Figure 1.2 Models of the minimal fusion machinery at three stages of the fusion process derived from different microscopy and crystallography techniques (I) Prefusion structures of SNAP25 and syntaxin 1A[11,23] inside the target membrane and synaptobrevin 2 inside the vesicular membrane.[24] (II) Models of the partly zippered trans-SNARE complex[20,23,24] anchored in both membranes and (III) the cis-SNARE complex after fusion with both TMDs inside one bilayer.[20] Modified according to Liang et al.[23]
Different mechanisms have been discussed of how exactly SNARE-zippering results in fusion pore formation and what the individual steps from vesicle docking until recycling are.[7,25,26] In the first scenario SNAREs play a direct role in fusion pore formation, as their TMDs line the fusion pore and connect the two lumen under exclusion of lipids.[27] However, this early idea proved to be unlikely due to the low number of SNAREs found to be necessary to drive membrane fusion.[28–30] In the second scenario the close proximity of the two bilayers after SNARE-zippering leads to lipid splaying and the formation of a lipidic fusion stalk with lifetimes of < 1 ms (Figure 1.3).[31,32] The stalk might enlarge into a hemifusion intermediate where only the lipids of the two outer leaflets of the opposing membranes are mixed.[33,34]
Whether this hemifusion state is a stable intermediate and when exactly an aqueous fusion pore is formed is, however, of constant debate.[33,35–37] After a first aqueous connection the fusion
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pore can expand and the vesicle collapses into the target membrane. This classic fusion pathway is often referred to as full-collapse fusion (FC) and is accompanied by clathrin-mediated endocytosis and recycling of vesicles in endosomes or inside the cell plasma.[7] However, an alternative fusion mode called kiss-and-run (KR) exocytosis involves a fusion pore that rapidly closes again before the vesicle collapses into the target membrane.[38] In that way the shape of the liposome, also referred to as -shape, remains intact and proteins as well as lipid material are preserved. Subsequently, the vesicle is directly retrieved from the plasma membrane in a fast endocytosis mode.[26] It is thought that KR is the dominant fusion mode at low frequency stimulation, however, the underlying mechanisms are still elusive and the relevance of KR is unclear.[38–41]
The large number of different proteins involved in these processes makes it difficult to unravel the molecular mechanisms that influence the fusion process in vivo. Consequently, it became obvious that in vitro studies are crucial to dissect the individual steps in the life cycle of the synaptic vesicle and to investigate the role of certain proteins, lipids, and other factors in a well-defined environment.
Figure 1.3 Model of a possible fusion pathway in SNARE-mediated membrane fusion. After vesicle docking and SNARE-zippering a fusion stalk is formed that evolves into a hemifusion intermediate. From this stable intermediate a fusion pore is formed, neurotransmitters are released, and the subsequent expansion of the pore leads to complete vesicle collapse.