VAMP Family Members
VAMP1: Vesicle-Associated Membrane Protein 1, Synaptobrevin 1, Syb1, Syb-1, VAMP-1, DKFZp686H12131
VAMP2: Vesicle-Associated Membrane Protein 2, Synaptobrevin 2, Syb2, Syb-2, VAMP-2, FLJ11460
VAMP3: Vesicle-Associated Membrane Protein 3, Cellubrevin, Cb, Ceb, VAMP-3, Synaptobrevin 3, Syb3
VAMP7: Vesicle-Associated Membrane Protein 7, Tetanus neurotoxin Insensitive Vesicle-Associated Membrane Protein (TI-VAMP, TIVAMP, Ti-VAMP), Synaptobrevin-like protein1, Synaptobrevin-like 1, Sybl1, VAMP-7, FLJ53045, FLJ53762, FLJ54296
Historical Background: The SNAREs Discovery
VAMP stands for Vesicle-Associated Membrane Protein (For reviews see (Jahn and Scheller 2006)). Nine VAMPs have been described: VAMPs 1, 2, 3, 4, 5, 7, 8, Ykt6, and Sec22b. The first discovered were VAMPs 1 and 2 initially identified as synaptic vesicle proteins thus also called Synaptobrevins 1 and 2. They were then found to be substrates of clostridial neurotoxins: botulinum neurotoxins B, D, F, and G and tetanus neurotoxin. Major breakthrough to define VAMP’s function originated from in vitro experiments aimed at characterizing transport between Golgi membranes (Balch et al. 1984). Indeed, fusion of Golgi vesicles with acceptor membranes is inhibited by pretreatment of cytosol with the cysteine alkylating agent NEM (N-ethylmaleimide) (Orci et al. 1989). The NEM-sensitive cytosolic component, essential for intra-Golgi transport after coat recruitment and vesicle formation, was identified as NSF protein (for NEM Sensitive Factor), the mammalian homologue of Sec18p identified by Novick and Schekman (Block and Rothman 1992). Whereas NSF could be found associated with membrane, purified NSF would not bind to Golgi membranes unless cytosol was added. This triggered the search and purification of the Soluble NSF Attachment Proteins or SNAPs. The homologous proteins α-, β-, and γ-SNAP were purified from bovine brain based on their ability to attach NSF to Golgi membranes and thus restore transport in the in vitro assay. As with NSF, a yeast mutant (Sec17) also identified in the screen of Novick and Schekman failed to restore SNAP activity, but could be rescued by addition of purified mammalian SNAP further strengthening the view that membrane fusion depended on a conserved fusion machinery. Consequently, three proteins, which had been previously cloned and found to be associated with the presynaptic terminal, were isolated from bovine brain as SNAP receptors: VAMP/Synaptobrevin, Syntaxin 1, and SNAP25. Further characterization of the complex formed by VAMP/Synaptobrevin, Syntaxin 1, and SNAP25 revealed that all three SNAP receptors or “SNARE” proteins are present in a single 20S particle together with NSF and SNAP, and that the ATPase activity of NSF dissociates the complex. An essential role of Synaptobrevin in neurotransmitter release was proposed simultaneously by demonstrating that Synaptobrevin is the target of several neurotoxins. Indeed, clostridial neurotoxins, the most potent blockers of neurotransmitter release, are proteases which cleave the synaptic SNARE proteins Synaptobrevin, SNAP25, and/or Syntaxin 1. Taking all these observations into consideration, Rothman and colleagues suggested a molecular mechanism, which would explain vesicle docking and fusion in molecular terms, the so-called SNARE hypothesis (Fig. 1a) (Rothman and Warren 1994; Söllner et al. 1993a, b): It is now well known that each transport step within the eukaryotic cell is mediated by a SNARE complex composed of one protein in the vesicle (the v-SNARE also called VAMP) and several in the target membrane (the t-SNAREs). These now historical findings suggested an important role for SNARE proteins at a late step in membrane fusion, a conclusion which has now been largely validated by a large series of experiments in vitro and in vivo and led to the award of the Nobel Prize in Physiology or Medicine to James E Rothman, Randy Sheckman, and Thomas Südhof. Indeed, SNARE-mediated lipid mixing can be efficiently recapitulated in vitro in assays reconstituting fusion with v-SNARE and t-SNARE proteins into separate liposome populations. These assays allowed for identification of lipidic and proteic regulations such as Sec1/Munc18 family of proteins (for a summary of some important results obtained with these in vitro experiments see Ji et al. (2010)).
Structure and Function
All SNAREs (v-and t-) share a segment in their cytosolic domain called the SNARE motif which can be considered the signature of these proteins (for reviews see Proux-Gillardeaux et al. (2005b); Jahn and Scheller (2006)). It consists of a domain of 60–70 amino acids (Fig. 1b) that is unstructured in solution but yet capable to adopt an α-helicoidal structure upon assembly into four-helix bundles when forming SNARE complexes with t-SNAREs.
Membrane fusion requires the assembly of one to five SNARE complexes Mohrmann et al. (2010); van den Bogaart et al. (2010), which are composed of one SNARE motif provided by the donor membrane v-SNARE and three SNARE motifs provided by two or three acceptor membrane t-SNAREs. In the best so far characterized SNARE complex one α-helix is contributed by the v-SNARE VAMP2, one by Syntaxin 1 and two are contributed by SNAP25. Hence, the SNARE core complex consists of four entwisted alpha-helices in which the “a” and “d” positions of the SNARE heptad repeats form a 16-layered hydrophobic core with a conserved ionic layer present at the center. This layer, also called the “zero” layer, is composed of one arginine residue (R), contributed by the v-SNARE, forming hydrogen bonds with three glutamines (Q), each provided by t-SNAREs. This results in another classification of the v- and t-SNAREs as R- and Q-SNAREs, respectively.
The assembly of v- and t-SNAREs into “trans” complexes bridges the opposing lipid bilayers of membranes belonging to donor and acceptor compartments, bringing them in proximity by “zippering” of the coiled-coil domains and inducing their fusion (Li et al. 2007). Several proteins of other families interact with SNAREs and regulate their docking, SNARE complex assembly, then their disassembly, such as members of the SM family comprising the already mentioned Sec1/Munc18, Complexin, Synaptophysin, and Synaptotagmin (Gerst 2003; Sudhof 2013).
Posttranslational modifications have also been suggested to regulate the SNARE activity including palmitoylation or phosphorylation. It has been already well described for the synaptic t-SNAREs Syntaxin1 and SNAP25 (Gerst 2003) and SNAP-25 phosphorylation by PKA or PKC contributes differentially to the control of exocytosis in PC12 cells by regulating SNARE complex formation (Gao et al. 2016), but little is known on the potential regulation of nonsynaptic v-SNAREs by posttranslational modifications. Recently a site of phosphorylation by PKC present on the nonsynaptic VAMPs in their SNARE domain has been identified. Its phosphorylation on VAMP8 still allows vesicular docking but reduces fusogenic activity (Malmersjo et al. 2016). On the contrary, a c-Src kinase-dependent phosphorylation in the VAMP7 longin domain has been reported to increase VAMP7-dependent exocytosis (Burgo et al. 2013). Thus, phosphorylation of SNARE proteins could be a general mechanism to restrict when and how much cells secrete.
Nine v- /R-SNAREs have been identified in the human genome: VAMP1/Synaptobrevin1, VAMP2/Synaptobrevin2, VAMP3/Cellubrevin, VAMP4, VAMP5/Myobrevin, VAMP7/Synaptobrevin-like protein1/TI-VAMP, VAMP8/Endobrevin, Sec22b/ER-Golgi SNARE of 24kDa/ERS24, and Ykt6. v-SNAREs are small abundant C-terminally anchored type II membrane proteins (Fig. 1b). They vary in their structure and size extension in their amino-terminus domain as detailed afterwards in the VAMP7 paragraph. The membrane anchor of VAMPs is a transmembrane domain except for Ykt6 which is anchored by a lipidic tail. Even if the different v-SNAREs share a high level of homology and display conserved structural and functional properties, they also have specific characteristics. Notably, the different VAMPs show different membrane fusion efficiency in cell fusion assay. VAMPs 1 and 3 being the most efficient, whereas VAMPs 4, 7, and 8 exhibit 30–50% lower fusion efficiency (Hasan et al. 2010).
VAMPs 1 and 2, also known as Synaptobrevins 1 and 2, are preferentially expressed in the brain where they are associated with synaptic vesicles and implicated in neurotransmitter release but also in secretory granules of endocrine and exocrine cells (Trimble 1993). Despite high homology, they show a differential distribution in rat brain, with partial overlap. The same has been described among the synapses of the mouse retina (Sherry et al. 2003). In both cases, VAMP2 is the most abundant and is widely distributed whereas VAMP1 is less abundant and more limited but it is the main isoform in certain brain areas (e.g., zona incerta of the subthalamus or nerve terminals surrounding thalamic neurons) (Raptis et al. 2005). Their localization can also vary locally during development as described in the mature versus developing deep cerebellar nuclei (Manca et al. 2014) as already described for the SNAP25a and b isoforms (Prescott and Chamberlain 2011). Thus differential expression of SNAREs could contribute to differential synaptic coding in the brain. VAMP1 is predominantly expressed in the spinal cord, particularly in motor neurons and motor nerve terminals of the neuromuscular junction where it plays an important role in Ca2+-triggered neurotransmitter release at the neuromuscular junction, the synaptic connection between nerve and muscle (Liu et al. 2011).
Astrocytes, the most abundant glial cells in the central nervous system and microglia another important constituent of glia, also express VAMP2 but there are discrepancies between astrocytes in acute brain slices, acutely dissociated astrocytes, and cultured astrocytes. There seems to be a better consensus about expression and function of VAMP3 in astrocytes (Ropert et al. 2016).
VAMPs 1/2, albeit primarily neuronal, are also expressed by certain non-neuronal cells in which they are involved in various regulated secretory processes. For example, VAMP2 is associated with insulin secretory granules in pancreatic beta cells in the islets of Langerhans and mediates insulin release (Hou et al. 2009). Interestingly, as for neurotransmitter release, insulin secretion is directly triggered by Ca2+-dependent secretory granule fusion with the plasma membrane.
VAMP3 was originally described as the nonneuronal isoform of VAMP2, with a widespread tissue distribution, and it participates to regulated and constitutive exocytosis. It has been localized to early endosomes.
VAMP3 has been involved in several important processes comprising polarized secretion, recycling of plasma membrane receptors, cell adherence, migration, degradation of extracellular matrix, and invasion. It has also been implicated in autophagy, regulating amphisome formation by controlling the fusion between multivesicular bodies, and autophagosomes (Fader et al. 2009).
VAMP3 is detected in the nervous system, where it is expressed mainly by glia and vascular cells. It has been implicated in CNS myelination by oligodendrocytes (Feldmann et al. 2011). VAMP3 is also expressed by Cajal Retzius cells (CRs), the first glutamatergic neurons to be born in the embryonic mouse cerebral cortex. VAMP3 modulates CRs migration speed and distribution in specific embryonic cortical territories (Barber et al. 2015).
Clostridial neurotoxins (tetanus neurotoxin (TeNT) and botulinum neurotoxins (BoNT) B, D, F, and G) cleave VAMPs 1, 2, and 3. Clostridial neurotoxins block neurotransmitter release in vivo and in cultured neurons. TeNT-expressing bipolar cell axons further show synapse formation defects in the retina (Kerschensteiner et al. 2009). Evoked release was completely abolished in dissociated hippocampal neurons expressing TeNT (Ben Fredj et al. 2010). In nonneuronal cells (Proux-Gillardeaux and Galli 2008), TeNT inhibits the recycling of plasma membrane receptors including: transferrin receptor (Galli et al. 1994), T-cell receptors to the immune synapse (Das et al. 2004), and integrin β1 in epithelial cells (Hager et al. 2010; Proux-Gillardeaux et al. 2005a; Skalski and Coppolino 2005; Skalski et al. 2010; Tayeb et al. 2005); it inhibits retrograde transport from early endosomes to the Golgi apparatus (Mallard et al. 2002), apical transport of H+-ATPase in kidney epithelial cells, and cytokine secretion (Murray et al. 2005), and it decreases the efficiency of phagocytosis by inhibiting focal exocytosis at sites of phagocytosis in macrophages, as does botulinum neurotoxin B (Hackam et al. 1998).
VAMP2-null mice show an almost complete block of neurotransmitter release leading to death at birth and a strong inhibition of synaptic vesicle endocytosis. It is unclear if neuronal synapses are fully normal in this mutant in vivo. However, using cortical explants from VAMP2 knockout mice and cortical explants treated with BoNT D, a role of VAMP2 during development was suggested. Indeed VAMP2 mediates axon-guidance through the trafficking of the Sema3A receptor constituted of Neuropilin1 and PlexinA1 (Zylbersztejn et al. 2012). Involvement of VAMP2 in neuronal development thus complements the data demonstrating his essential function in mediating neurotransmitter release in the adult nervous system.
Transgenic mice expressing clostridial neurotoxins (TeNT and iBot) allowed to put together the two previous pharmacological and genetic approaches. In the first, expression of the TeNT light chain is driven in a Tetracycline-dependent manner and leads to cleavage of vesicle-associated membrane protein 1-3, depending on which VAMP is expressed. In this model, reversible suppression of glutamatergic neurotransmission can be manipulated with spatiotemporal accuracy by DOX treatment and removal (Yamamoto et al. 2003). They referred to this novel gene-manipulating technique as a “reversible neurotransmission blocking (RNB).” This allowed them to show that conditioned eyeblink learning is formed and stored without cerebellar granule cell transmission (Wada et al. 2007).
In the second, expression of BoNT B light chain is driven in a Cre recombinase-dependent manner and leads also to the cleavage of the present VAMPs 1 to 3. Toxin-mediated elimination of these VAMPs in retinal glial cells inhibits vesicular glutamate release and impairs volume regulation in these cells, but does not affect retinal histology and visual processing (Slezak et al. 2012).
IBot mice and VAMP3 KO mice were used to demonstrate for the first time a role of VAMP3 in postmitotic neurons (Cajal Retzius cells) migration and in early cortical brain development (Barber et al. 2015). Previously, this v-SNARE was thought to be expressed exclusively in nonneuronal cells in the mature brain and was considered to have a redundant role with VAMPs 1 and 2 when expressed in the same cells. It now appears that VAMP3 is also expressed in transient neurons during development.
VAMP-3 depletion by RNA interference inhibits the delivery of tumor necrosis factor-α to the plasma membrane in macrophages (Kay et al. 2006; Murray et al. 2005). Silencing of VAMP3 by RNAi also results in a significant reduction in ciliary length revealing a role for this v-SNARE in the trafficking events that regulate the growth of this organelle in this process but also for the delivery of recycling T-cell receptors to immune synapse (Finetti et al. 2015).
In contrast to VAMPs 1, 2, and 3, VAMP7 is resistant to clostridial neurotoxins (tetanus and botulinum neurotoxins B, D, F, and G). For that reason VAMP7 was also called Tetanus neurotoxin-Insensitive Vesicle-Associated Membrane Protein (TI-VAMP). VAMP7 is encoded by Synaptobrevin-like 1 (Sybl1) gene, the first pseudoautosomal gene found to be X and Y inactivated. Sybl1 is ubiquitously expressed and highly conserved in eukaryotes (with the exception of yeast which does not carry the gene).
In adult rat brain, VAMP7 is localized in the somatodendritic compartment of neurons and also in subsets of axon terminals including, the hippocampal mossy fibers of the dentate gyrus and of CA3, the striatal peridendritic terminal plexuses in the globus pallidus, substantia nigra pars reticulata, peridendritic plexuses in the central nucleus of the amygdala, and the primary sensory afferents in the dorsal horn of the spinal cord (Muzerelle et al. 2003).
Unlike brevins (VAMPs 1, 2, 3, and 8), but like Syntaxins, VAMP7 possess a long amino-terminal extension of about 100 amino acids, also called the “Longin” domain (Fig. 1). Thus VAMP7 is seen as the prototype of the family of “Longin” v-SNAREs also containing Sec22b and Ykt6. Their Longin domain adopts a conserved globular “profilin-like” structure, similar to the profilin domain of the human SEDL protein and of domain present in the sigma and mu subunits of the AP-2 clathrin adaptor complex and other proteins (Daste et al. 2015). This domain adopts a closed conformation folded onto their SNARE domain, preventing the formation of SNARE complexes (Vivona et al. 2010). The Longin domain is necessary for proper targeting, because mutations or deletion of this domain result in mistargeting of VAMP7 to early endosomes (Martinez-Arca et al. 2003). More precisely, in VAMP7, the Longin domain has several functions: It inhibits the formation of SNARE complexes and it binds to the δ subunit of the molecular coat AP-3 protein to target VAMP7 to late endosomes or synaptic vesicles. Interestingly Dictostelium discoideum VAMP7 has been shown to interact with all four adaptor protein complexes (AP-1, 2, 3, and 4) and at least AP-2 and AP-3 participate in its sorting to the endocytic pathways. VAMP7 endocytosis also depends on the Longin domain. Indeed Hrb binds the Longin domain allowing VAMP7 recruitment into clathrin-coated vesicles and endocytosis (Chaineau et al. 2008; Pryor et al. 2008).
VAMP7 localizes to the trans-Golgi network (TGN) where it is the starting point of a molecular network that mediates its transport from the Golgi apparatus to the cell periphery and in particular to neurite tips. This network comprises Varp, a VAMP7 interactor, Rab21 GEF and partner of both GolginA4 and the kinesin1 Kif5A, and MACF1, a GolginA4 partner and Rab21 effector (Burgo et al. 2012). VAMP7 is also localized in late endosomes and lysosomes where it allows for heterotypic fusion between these compartments and lysosomal secretion, a process required for plasma membrane repair, cell migration, and metalloproteinase-dependent cell invasion (Proux-Gillardeaux et al. 2007; Rao et al. 2004; Steffen et al. 2008; Williams et al. 2014). VAMP7 is involved in fusion of secretory vesicles with the plasma membrane, which is necessary for neurite growth (Martinez-Arca et al. 2000), phagocytosis (Braun et al. 2004), lysosomal, and granule secretion in polarized, migrating, and invading cells. VAMP7 modulates also ciliary biogenesis in kidney cells (Szalinski et al. 2014). VAMP3 and VAMP7 are involved in specific autophagy steps and in the selection of the pathways leading to generation of ultrastructurally different LC3 compartments (Fader et al. 2009; Ligeon et al. 2014).VAMP7 is involved in phagophore formation, an early event in autophagosome biogenesis (Moreau et al. 2011) and later fusion with lysosomes (Fader et al. 2009).
In cultured neurons VAMP7-mediated exocytosis has been shown to mediate axonal and dendritic growth (Martinez-Arca et al. 2000) and is particularly active in early phases of axon formation (Gupton and Gertler 2010) and Netrin-1-dependent attraction (Cotrufo et al., 2011). In fact, it has recently been suggested that VAMP4 mediates early transport of IGF1R in the axon and that VAMP7 could subsequently be involved in axonal elongation (Grassi et al. 2015). This model (Wojnacki Fonseca and Galli 2016) is particularly appealing because IGF1 was shown to enhance VAMP7 exocytosis (Burgo et al. 2013). Later in development and adulthood, VAMP7 may be involved in neurotransmitter release in some specific neurons (Hua et al. 2011; Scheuber et al. 2006) and higher brain function (Danglot et al. 2012). VAMP7 mediates the transport of rafts and GPI-anchored proteins including the prion protein to the plasma membrane (Molino et al. 2015). VAMP7 function has been linked to immune responses, notably by controlling T cell activation (Larghi et al. 2013) and by regulating exocytic trafficking of Interleukin 12 in dendritic cells (Chiaruttini et al. 2016).
We apologize to all the authors that are not cited in the text due to strong reference limitation. Our work is supported in part by the Institut National de la Santé et de la Recherche Médicale (INSERM), and the Centre National de la Recherche Scientifique (CNRS) and grants from the Fondation pour la Recherche Médicale (FRM), the Association pour la Recherche sur le Cancer (ARC), and the Who am I? Labex (Idex ANR-11-IDEX-0005-01).
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