A method to detect binary interactions among SNAREs, membrane proteins mediating vesicle fusion, in Arabidopsis cells was established. In this method, a pair of recombinant SNAREs is first expressed within Arabidopsis protoplasts at levels similar to their endogenous proteins in 96-well plates. Changes of the interaction are then detected by luminescence. Here, we report that the interaction of SYP122 and VAMP721, a SNARE pair mediating exocytosis, is enhanced when Arabidopsis protoplasts are incubated in the dark. Microscopic observation of plants expressing GFP-SYP122 by the syp122 promoter suggests SYP122 is expressed in the root tip when the seedlings are grown in the dark but not in the light. In the identical dark-grown condition, the subcellular localization of SYP111/KNOLLE, specifically expressed in dividing cells, is altered. Together with our previous report, we hypothesize that expression, localization and interaction of SNAREs are selectively altered by light conditions to regulate cargo transports in Arabidopsis.
luciferase complementation assay; arabidopsis; SNARE; vesicle trafficking; light
SNAREs provide the specificity and energy for the fusion of vesicles with their target membrane, but how they are sorted into the appropriate vesicles on post-Golgi trafficking pathways is largely unknown. We demonstrate that the clathrin-mediated endocytosis of the SNARE VAMP7 is directly mediated by Hrb, a clathrin adaptor and ArfGAP. Hrb wraps 20 residues of its unstructured C-terminal tail around the folded VAMP7 longin domain, demonstrating that unstructured regions of clathrin adaptors can select cargo. Disrupting this interaction by mutation of the VAMP7 longin domain or depletion of Hrb causes VAMP7 to accumulate on the cell's surface. However, the SNARE helix of VAMP7 binds back onto its longin domain, outcompeting Hrb for binding to the same groove and suggesting that Hrb-mediated endocytosis of VAMP7 occurs only when VAMP7 is incorporated into a cis-SNARE complex. These results elucidate the mechanism of retrieval of a postfusion SNARE complex in clathrin-coated vesicles.
CELLBIO; SIGNALING; PROTEINS
Effective recognition of pathogens and rapid execution of immune responses are essential for the survival of living organisms. Cell-autonomous immune responses of animal and plant cells rely on pattern recognition receptors that can distinguish self from non-self structures and that are able to activate a molecular execution machinery that ultimately terminates most pathogen attacks. Reminiscent of the situation in mammalian T cells, accumulating evidence points to a key role of vesicle trafficking and exocytosis in plant innate immunity. In this context, our recent finding that ternary soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complexes comprising PEN1, SNAP33 and VAMP721/722 function at pathogen entry sites is instrumental in understanding the execution of plant immune responses at the cell periphery. Our study further revealed unexpected overlapping functions of the same SNARE complexes in disease resistance and development. Here, we discuss the potential identity of cargo delivered through the PEN1-SNAP33-VAMP721/722-dependent secretory pathway and the necessity for a tight regulation of SNARE complex formation to avoid unintentional release of toxic load.
PEN1; plant immunity; secretory pathway; SNARE; VAMP721/722
Intracellular vesicle fusion is mediated by the interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs). The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments. To fully understand the specific role of each VAMP in vesicle trafficking, it is important to determine if VAMPs have differential membrane fusion activities.
In this study, we developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of β-galactosidase, and examined fusogenic pairings between the seven VAMPs, i.e., VAMPs 1, 2, 3, 4, 5, 7 and 8, and two plasma membrane t-SNARE complexes, syntaxin1/SNAP-25 and syntaxin4/SNAP-25. VAMPs 1, 2, 3, 4, 7 and 8 drove fusion efficiently, whereas VAMP5 was unable to mediate fusion with the t-SNAREs. By expressing VAMPs 1, 3, 4, 7 and 8 at the same level, we further compared their membrane fusion activities. VAMPs 1 and 3 had comparable and the highest fusion activities, whereas VAMPs 4, 7 and 8 exhibited 30–50% lower fusion activities. Moreover, we determined the dependence of cell fusion activity on VAMP1 expression level. Analysis of the dependence data suggested that there was no cooperativity of VAMP proteins in the cell fusion reaction.
These data indicate that VAMPs have differential membrane fusion capacities, and imply that with the exception of VAMP5, VAMPs are essentially redundant in mediating fusion with plasma membrane t-SNAREs.
The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been demonstrated that clostridial neurotoxins (NTs) proteolyze the vesicle SNAREs (v-SNAREs) vesicle-associated membrane protein (VAMP)/brevins and their partners, the target SNAREs (t-SNAREs) syntaxin 1 and SNAP25. Yet, several exocytotic events, including apical exocytosis in epithelial cells, are insensitive to numerous clostridial NTs, suggesting the presence of SNARE-independent mechanisms of exocytosis. In this study we found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs. In epithelial cells, TI-VAMP–containing vesicles were concentrated in the apical domain, and the protein was detected at the apical plasma membrane by immunogold labeling on ultrathin cryosections. Syntaxin 3 and SNAP23 were codistributed at the apical plasma membrane where they formed NEM-dependent SNARE complexes with TI-VAMP and cellubrevin. We suggest that TI-VAMP, SNAP23, and syntaxin 3 can participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specific exocytosis in clostridial NT-resistant pathways.
Vesicle-associated-membrane protein 8 (VAMP8) is highly expressed in the kidney, but the exact physiological and molecular functions executed by this v-SNARE protein in nephrons remain elusive. Here, we show that the depletion of VAMP8 in mice resulted in hydronephrosis. Furthermore, the level of the vasopressin-responsive water channel aquaporin 2 (AQP2) was increased by three- to fivefold in VAMP8-null mice. Forskolin and [desamino-Cys1, D-Arg8]-vasopressin (DDAVP)-induced AQP2 exocytosis was impaired in VAMP8-null collecting duct cells. VAMP8 was revealed to colocalize with AQP2 on intracellular vesicles and to interact with the plasma membrane t-SNARE proteins syntaxin4 and syntaxin3, suggesting that VAMP8 mediates the regulated fusion of AQP2-positive vesicles with the plasma membrane.
VAMP7 is involved in the fusion of late endocytic compartments with other membranes. One possible mechanism of VAMP7 delivery to these late compartments is via the AP3 trafficking adaptor. We show that the linker of the δ-adaptin subunit of AP3 binds the VAMP7 longin domain and determines the structure of their complex. Mutation of residues on both partners abolishes the interaction in vitro and in vivo. The binding of VAMP7 to δ-adaptin requires the VAMP7 SNARE motif to be engaged in SNARE complex formation and hence AP3 must transport VAMP7 when VAMP7 is part of a cis-SNARE complex. The absence of δ-adaptin causes destabilization of the AP3 complex in mouse mocha fibroblasts and mislocalization of VAMP7. The mislocalization can be rescued by transfection with wild-type δ-adaptin but not by δ-adaptin containing mutations that abolish VAMP7 binding, despite in all cases intact AP3 being present and LAMP1 trafficking being rescued.
► AP3-containing vesicles transport VAMP7 and LAMP1 from early to late endosomes ► The ear/trunk linker of the AP3 δ-subunit binds in a groove on a VAMP7 longin domain ► Binding of VAMP by AP3 can only occur when VAMP is part of a cis-SNARE complex ► VAMP7 is not required for the fusion of AP3-containing vesicles with late endosomes
VAMP7 SNARE complexes on late endosomes determine the specificity of membrane fusion there. But how is VAMP7 localization maintained? Kent et al. define the structure of and requirement for VAMP7 interactions with the cargo adaptor AP3. AP3 uses distinct surfaces for VAMP7 versus other cargoes to recycle postfusion cis-SNARE complexes.
Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of α-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the N-terminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the N-terminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins.
SNARE; VAMP3; membrane fusion; vesicular trafficking
SNARE (soluble-N-ethylmaleimide-sensitive factor attachment receptor) proteins mediate the recognition and fusion of transport vesicles in eukaryotic cells. The SNARE protein VAMP8 (also called endobrevin) is involved in the fusion of late endosomes and in some pathways of regulated exocytosis. In a subset of mice deficient for the SNARE protein VAMP8, a severe alteration of the thymus and in T lymphocyte development was observed and characterized. The size of the thymus and the number of thymocytes were dramatically reduced compared with those in heterozygous littermates. Further, the compartmentalization into cortex and medulla and the organization of the thymus epithelium were disturbed. The numbers of all thymocyte subpopulations were reduced, with the CD4 and CD8 double-positive thymocytes being most severely affected. The proportion of proliferating thymocytes was reduced, and the staining of apoptotic cells in situ and ex vivo indicated an increased number of apoptotic cells. Isolated thymocytes of Vamp8−/− mice were more susceptible to various apoptotic stimuli including glucocorticoids, FAS receptor, and CD3/CD28-mediated signaling in vitro, even before an increased number of apoptotic cells was detectable in situ. However, bone marrow of phenotypically affected Vamp8−/− mice was readily able to repopulate immunodeficient hosts suggesting that the SNARE protein VAMP8 has a specific function in the thymic stroma affecting the proliferation and apoptosis of T lymphocytes during maturation in the thymus.
SNARE; T-cell; Thymus; Apoptosis; Knockout mouse
Screening of a library derived from primary human endothelial cells
revealed a novel human isoform of vesicle-associated membrane protein-1
(VAMP-1), a protein involved in the targeting and/or fusion of
transport vesicles to their target membrane. We have termed this novel
isoform VAMP-1B and designated the previously described isoform
VAMP-1A. VAMP-1B appears to be an alternatively spliced form of VAMP-1.
A similar rat splice variant of VAMP-1 (also termed VAMP-1B) has
recently been reported. Five different cultured cell lines, from
different lineages, all contained VAMP-1B but little or no detectable
VAMP-1A mRNA, as assessed by PCR. In contrast, brain mRNA contained
VAMP-1A but no VAMP-1B. The VAMP-1B sequence encodes a protein
identical to VAMP-1A except for the carboxy-terminal five amino acids.
VAMP-1 is anchored in the vesicle membrane by a carboxy-terminal
hydrophobic sequence. In VAMP-1A the hydrophobic anchor is followed by
a single threonine, which is the carboxy-terminal amino acid. In
VAMP-1B the predicted hydrophobic membrane anchor is shortened by four
amino acids, and the hydrophobic sequence is immediately followed by
three charged amino acids, arginine-arginine-aspartic acid.
Transfection of human endothelial cells with epitope-tagged VAMP-1B
demonstrated that VAMP-1B was targeted to mitochondria whereas VAMP-1A
was localized to the plasma membrane and endosome-like structures.
Analysis of C-terminal mutations of VAMP-1B demonstrated that
mitochondrial targeting depends both on the addition of positive charge
at the C terminus and a shortened hydrophobic membrane anchor. These
data suggest that mitochondria may be integrated, at least at a
mechanistic level, to the vesicular trafficking pathways that govern
protein movement between other organelles of the cell.
VAMP/synaptobrevin is part of the synaptic vesicle docking and fusion complex and plays a central role in neuroexocytosis. Two VAMP (vesicle- associated membrane protein) isoforms are expressed in the nervous system and are differently distributed among the specialized parts of the tissue. Here, VAMP-1 and -2 are shown to be present in all rat tissues tested, including kidney, adrenal gland, liver, pancreas, thyroid, heart, and smooth muscle. The two isoforms are differentially expressed in various tissues and their level may depend on differentiation. VAMP-1 is restricted to exocrine pancreas and to kidney tubular cells, whereas VAMP-2 is the predominant isoform present in Langerhans islets and in glomerular cells. Both isoforms show a patchy vesicular intracellular distribution in confocal microscopy. The present results provide evidence for the importance of neuronal VAMP proteins in the physiology of all cells.
SNAREs provide a large part of the specificity and energy needed for membrane fusion and, to do so, must be localized to their correct membranes. Here, we show that the R-SNAREs VAMP8, VAMP3, and VAMP2, which cycle between the plasma membrane and endosomes, bind directly to the ubiquitously expressed, PtdIns4,5P2-binding, endocytic clathrin adaptor CALM/PICALM. X-ray crystallography shows that the N-terminal halves of their SNARE motifs bind the CALMANTH domain as helices in a manner that mimics SNARE complex formation. Mutation of residues in the CALM:SNARE interface inhibits binding in vitro and prevents R-SNARE endocytosis in vivo. Thus, CALM:R-SNARE interactions ensure that R-SNAREs, required for the fusion of endocytic clathrin-coated vesicles with endosomes and also for subsequent postendosomal trafficking, are sorted into endocytic vesicles. CALM's role in directing the endocytosis of small R-SNAREs may provide insight into the association of CALM/PICALM mutations with growth retardation, cognitive defects, and Alzheimer's disease.
► Binding to CALM selects VAMPs 8, 3, and 2 for incorporation into endocytic CCVs ► The CALM ANTH domain binds VAMPs and PtdIns4,5P2 simultaneously ► Helical N-terminal halves of VAMP SNARE motifs displace the CALM ANTH final helix ► VAMP endocytosis is blocked by mutation of residues in the CALM:SNARE interface
CALM recognizes the SNARE motif of small R-SNARE proteins as a sorting signal to direct R-SNARE endocytosis and trafficking to the appropriate intracellular compartment while simultaneously shielding the SNARE motif from inappropriate interactions. This unique role for CALM, distinct from other clathrin adaptors, may explain the genetic association of the CALM/PICALM gene with neurological disorders.
Helicobacter pylori-produced cytotoxin VacA induces intracellular vacuolation. The VacA-induced vacuole is assumed to represent the pathological status of intracellular trafficking. The fusion mechanism of the endosomes requires the formation of a tight complex between the Q-SNAREs and the R-SNAREs. We recently reported that syntaxin 7, a family member of the Q-SNARE protein, is involved in VacA-induced vacuole formation. In order to further elucidate the molecular mechanism, we identified the participation of vesicle-associated membrane protein 7 (VAMP7) as a partner of syntaxin 7. Immunocytochemistry revealed endogenous VAMP7 to be localized to the vacuoles induced by VacA. A Northern blotting study demonstrated that VacA intoxication increased VAMP7 mRNA in a time-dependent manner. VAMP7 was coimmunoprecipitated with syntaxin 7, and the amounts of endogenous VAMP7 and syntaxin 7 bound to syntaxin 7 and VAMP7, respectively, increased in response to VacA. The down-regulation of VAMP7 using small interfering RNA inhibited VacA-induced vacuolation, and the transient transfection of dominant-negative mutant VAMP7, the N-terminal domain of VAMP7, also inhibited the vacuolation. We therefore conclude that R-SNARE VAMP7 plays an important role in VacA-induced vacuolation as a partner of Q-SNARE syntaxin 7.
Like neuronal synaptic vesicles, intracellular GLUT4-containing vesicles must dock and fuse with the plasma membrane, thereby facilitating insulin-regulated glucose uptake into muscle and fat cells. GLUT4 colocalizes in part with the vesicle SNAREs VAMP2 and VAMP3. In this study, we used a single-cell fluorescence-based assay to compare the functional involvement of VAMP2 and VAMP3 in GLUT4 translocation. Transient transfection of proteolytically active tetanus toxin light chain cleaved both VAMP2 and VAMP3 proteins in L6 myoblasts stably expressing exofacially myc-tagged GLUT4 protein and inhibited insulin-stimulated GLUT4 translocation. Tetanus toxin also caused accumulation of the remaining C-terminal VAMP2 and VAMP3 portions in Golgi elements. This behavior was exclusive to these proteins, because the localization of intracellular myc-tagged GLUT4 protein was not affected by the toxin. Upon cotransfection of tetanus toxin with individual vesicle SNARE constructs, only toxin-resistant VAMP2 rescued the inhibition of insulin-dependent GLUT4 translocation by tetanus toxin. Moreover, insulin caused a cortical actin filament reorganization in which GLUT4 and VAMP2, but not VAMP3, were clustered. We propose that VAMP2 is a resident protein of the insulin-sensitive GLUT4 compartment and that the integrity of this protein is required for GLUT4 vesicle incorporation into the cell surface in response to insulin.
Platelet secretion is critical to hemostasis. Release of granular cargo is mediated by soluble NSF attachment protein receptors (SNAREs), but despite consensus on t-SNAREs usage, it is unclear which Vesicle Associated Membrane Protein (VAMPs: synaptobrevin/VAMP-2, cellubrevin/VAMP-3, TI-VAMP/VAMP-7, and endobrevin/VAMP-8) is required. We demonstrate that VAMP-8 is required for release from dense core granules, alpha granules, and lysosomes. Platelets from VAMP-8−/− mice have a significant defect in agonist-induced secretion, though signaling, morphology, and cargo levels appear normal. In contrast, VAMP-2+/−, VAMP-3−/−, and VAMP-2+/−/VAMP-3−/− platelets showed no defect. Consistently, tetanus toxin had no effect on secretion from permeabilized mouse VAMP-3−/− platelets or human platelets, despite cleavage of VAMP-2 and/or -3. Tetanus toxin does block the residual release from permeabilized VAMP-8−/− platelets, suggesting a secondary role for VAMP-2 and/or -3. These data imply a ranked redundancy of v-SNARE usage in platelets and suggest that VAMP-8−/− mice will be a useful in vivo model to study platelet exocytosis in hemostasis and vascular inflammation.
SNAREs provide energy and specificity to membrane fusion events. Fusogenic trans-SNARE complexes are assembled from Q-SNAREs embedded in one membrane and an R–SNARE embedded in the other. Regulation of membrane fusion events is crucial for intracellular trafficking. We identify the endosomal protein Varp as an R-SNARE-binding regulator of SNARE complex formation. Varp co-localises with and binds to VAMP7, an R-SNARE involved in both endocytic and secretory pathways. We present the structure of the second ankyrin repeat domain of mammalian Varp in complex with the cytosolic portion of VAMP7. The VAMP7 SNARE motif is trapped between Varp and the VAMP7 longin domain and hence Varp kinetically inhibits VAMP7’s ability to form SNARE complexes. This inhibition will be increased when Varp can also bind to other proteins present on the same membrane as the VAMP7 such as Rab32:GTP.
How vesicular transport participates in neurite outgrowth is still poorly understood. Neurite outgrowth is not sensitive to tetanus neurotoxin thus does not involve synaptobrevin-mediated vesicular transport to the plasma membrane of neurons. Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is a vesicle-SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment protein [SNAP] receptor), involved in transport to the apical plasma membrane in epithelial cells, a tetanus neurotoxin-resistant pathway. Here we show that TI-VAMP is essential for vesicular transport-mediating neurite outgrowth in staurosporine-differentiated PC12 cells. The NH2-terminal domain, which precedes the SNARE motif of TI-VAMP, inhibits the association of TI-VAMP with synaptosome-associated protein of 25 kD (SNAP25). Expression of this domain inhibits neurite outgrowth as potently as Botulinum neurotoxin E, which cleaves SNAP25. In contrast, expression of the NH2-terminal deletion mutant of TI-VAMP increases SNARE complex formation and strongly stimulates neurite outgrowth. These results provide the first functional evidence for the role of TI-VAMP in neurite outgrowth and point to its NH2-terminal domain as a key regulator in this process.
membrane traffic; neurite outgrowth; SNAREs; TI-VAMP; SNAP25
Acrosomal exocytosis (AE) is an intracellular multipoint fusion reaction of the sperm plasma membrane (PM) with the outer acrosomal membrane (OAM). This unique exocytotic event enables the penetration of the sperm through the zona pellucida of the oocyte. We previously observed a stable docking of OAM to the PM brought about by the formation of the trans-SNARE complex (syntaxin 1B, SNAP 23 and VAMP 3). By using electron microscopy, immunochemistry and immunofluorescence techniques in combination with functional studies and proteomic approaches, we here demonstrate that calcium ionophore-induced AE results in the formation of unilamellar hybrid membrane vesicles containing a mixture of components originating from the two fused membranes. These mixed vesicles (MV) do not contain the earlier reported trimeric SNARE complex but instead possess a novel trimeric SNARE complex that contained syntaxin 3, SNAP 23 and VAMP 2, with an additional SNARE interacting protein, complexin 2. Our data indicate that the earlier reported raft and capacitation-dependent docking phenomenon between the PM and OAM allows a specific rearrangement of molecules between the two docked membranes and is involved in (1) recruiting SNAREs and complexin 2 in the newly formed lipid-ordered microdomains, (2) the assembly of a fusion-driving SNARE complex which executes Ca2+-dependent AE, (3) the disassembly of the earlier reported docking SNARE complex, (4) the recruitment of secondary zona binding proteins at the zona interacting sperm surface. The possibility to study separate and dynamic interactions between SNARE proteins, complexin and Ca2+ which are all involved in AE make sperm an ideal model for studying exocytosis.
Natural killer (NK) cells eliminate cancer and virus-infected cells through cytolytic activity. The last step in NK cell cytotoxicity, resulting in exocytosis of granule content, requires fusion of lytic granules with the plasma membrane. Proteins from the SNARE family mediate membrane fusion events in the cell. Here we show that NK cells express all members of the R-SNARE subgroup. Two of these R-SNARE proteins, VAMP4 and VAMP7, co-localize with lytic granules during cytotoxic interactions. However, only VAMP7 associates with perforin-containing granules in non-activated cells, indicating that the two VAMPs have different functions in exocytosis. Using both the tumor NK cell line, YTS, and peripheral NK cells we show that disruption of expression of either VAMP4 or VAMP7 inhibits release of lytic granules and severely impairs NK cell cytotoxic activity. Furthermore, VAMP7 but not VAMP4 is involved in IFNγ secretion in NK cells, indicating that VAMP7 is involved in many fusion processes and thus plays a more general function in NK cell activity than VAMP4.
lytic granules; perforin; vesicle fusion; immunological synapse
The control of intracellular vesicle trafficking is an ideal target to weigh the role of alternative splicing in shaping genomes to make cells. Alternative splicing has been reported for several Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptors of the vesicle (v-SNAREs) or of the target membrane (t-SNARES), which are crucial to intracellular membrane fusion and protein and lipid traffic in Eukaryotes. However, splicing has not yet been investigated in Longins, i.e. the most widespread v-SNAREs. Longins are essential in Eukaryotes and prototyped by VAMP7, Sec22b and Ykt6, sharing a conserved N-terminal Longin domain which regulates membrane fusion and subcellular targeting. Human VAMP7/TI-VAMP, encoded by gene SYBL1, is involved in multiple cell pathways, including control of neurite outgrowth.
Alternative splicing of SYBL1 by exon skipping events results in the production of a number of VAMP7 isoforms. In-frame or frameshift coding sequence modifications modulate domain architecture of VAMP7 isoforms, which can lack whole domains or domain fragments and show variant or extra domains. Intriguingly, two main types of VAMP7 isoforms either share the inhibitory Longin domain and lack the fusion-promoting SNARE motif, or vice versa. Expression analysis in different tissues and cell lines, quantitative real time RT-PCR and confocal microscopy analysis of fluorescent protein-tagged isoforms demonstrate that VAMP7 variants have different tissue specificities and subcellular localizations. Moreover, design and use of isoform-specific antibodies provided preliminary evidence for the existence of splice variants at the protein level.
Previous evidence on VAMP7 suggests inhibitory functions for the Longin domain and fusion/growth promoting activity for the Δ-longin molecule. Thus, non-SNARE isoforms with Longin domain and non-longin SNARE isoforms might have somehow opposite regulatory functions. When considering splice variants as "natural mutants", evidence on modulation of subcellular localization by variation in domain combination can shed further light on targeting determinants. Although further work will be needed to characterize identified variants, our data might open the route to unravel novel molecular partners and mechanisms, accounting for the multiplicity of functions carried out by the different members of the Longin proteins family.
SNARE protein complexes are key mediators of exocytosis by juxtaposing opposing membranes, leading to membrane fusion. SNAREs generally consist of one or two core domains that can form a four-helix bundle with other SNARE core domains. Some SNAREs, such as syntaxin target-SNAREs and longin vesicular-SNAREs, have independent, folded N-terminal domains that can interact with their respective SNARE core domains and thereby affect the kinetics of SNARE complex formation. This autoinhibition mechanism is believed to regulate the role of the longin VAMP7/TI-VAMP in neuronal morphogenesis. Here we use nuclear magnetic resonance spectroscopy to study the longin-SNARE core domain interaction for VAMP7. Using complete backbone resonance assignments, chemical shift perturbations analysis, and hydrogen/deuterium exchange experiments, we conclusively show that VAMP7 adopts a preferentially closed conformation in solution. Taken together, the closed conformation of longins is conserved, in contrast to the syntaxin family of SNAREs for which mixtures of open and closed states have been observed. This may indicate different regulatory mechanisms for SNARE complexes containing syntaxins and longins, respectively.
Membrane Fusion; Neurodevelopment; NMR; Protein Conformation; Vesicles; Longin; Synaptobrevin; Syntaxin
Synaptic vesicles in the brain harbor several SNARE proteins. With the exception of synaptobrevin2/VAMP2 (syb2) that is directly involved in vesicle fusion, the role of these SNAREs in neurotransmission is unclear. Here, we show that in mice while syb2 drives rapid Ca2+-dependent synchronous neurotransmission, the structurally homologous SNARE protein VAMP4 selectively maintains bulk Ca2+-dependent asynchronous release. At inhibitory nerve terminals, up- or down-regulation of VAMP4 causes a correlated change in asynchronous release. Biochemically, VAMP4 forms a stable complex with SNAREs syntaxin-1 and SNAP-25 that does not interact with complexins or synaptotagmin-1, proteins essential for synchronous neurotransmission. Optical imaging of individual synapses indicates that VAMP4 and syb2 trafficking show minimal overlap. Taken together, these findings suggest that VAMP4 and syb2 diverge functionally, traffic independently and support distinct forms of neurotransmission. These results provide molecular insight into how synapses diversify their release properties by taking advantage of distinct synaptic vesicle-associated SNAREs.
Integrins are transmembrane receptors for cell adhesion to the extracellular matrix. In cell migration, integrins are endocytosed from the plasma membrane or the cell surface, transported in vesicles and exocytosed actively at the cell front. In the current study, we examined the roles of VAMP3, a SNARE protein that mediates exocytosis, in cell migration and integrin trafficking. Small interfering RNA (siRNA)-induced silencing of VAMP3 inhibited chemotactic cell migration by more than 60% without affecting cell proliferation. VAMP3 silencing reduced the levels of β1 integrin at the cell surface but had no effect on total cellular β1 integrin, indicating that VAMP3 is required for trafficking of β1 integrin to the plasma membrane. Furthermore, VAMP3 silencing diminished cell adhesion to laminin but not to fibronectin or collagen. Taken together, these data suggest that VAMP3-dependent integrin trafficking is crucial in cell migration and cell adhesion to laminin.
SNARE; VAMP3; cell migration; integrin; cell adhesion
Renin is essential for blood pressure control. Renin is stored in granules in juxtaglomerular (JG) cells, located in the pole of the renal afferent arterioles. The second messenger cAMP stimulates renin release. However, it is unclear whether fusion and exocytosis of renin-containing granules is involved. In addition, the role of the fusion proteins, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment proteins), in renin release from JG cells has not been studied. The vesicle SNARE proteins VAMP2 (vesicle associated membrane protein 2) and VAMP3 mediate cAMP-stimulated exocytosis in other endocrine cells. Thus, we hypothesized that VAMP2 and/or -3 mediate cAMP-stimulated renin release from JG cells. By fluorescence-activated cell sorting, we isolated JG cells expressing green fluorescent protein and compared the relative abundance of VAMP2/3 in JG cells versus total mouse kidney mRNA by quantitative PCR. We found that VAMP2 and VAMP3 mRNA are expressed and enriched in JG cells. Confocal imaging of primary cultures of JG cells showed that VAMP2 (but not VAMP3) co-localized with renin-containing granules. Cleavage of VAMP2 and VAMP3 with tetanus toxin blocked cAMP-stimulated renin release from JG cells by ∼50% and impaired cAMP-stimulated exocytosis by ∼50%, as monitored with FM1–43. Then we specifically knocked down VAMP2 or VAMP3 by adenoviral-mediated delivery of short hairpin silencing RNA. We found that silencing VAMP2 blocked cAMP-induced renin release by ∼50%. In contrast, silencing VAMP3 had no effect on basal or cAMP-stimulated renin release. We conclude that VAMP2 and VAMP3 are expressed in JG cells, but only VAMP2 is targeted to renin-containing granules and mediates the stimulatory effect of cAMP on renin exocytosis.
Cyclic AMP (cAMP); Fusion Protein; Kidney; Renal Physiology; Renin; Hypertension; SNAREs
After synaptic vesicles fuse with the plasma membrane and release their contents, vesicle membrane proteins recycle by endocytosis and are targeted to newly formed synaptic vesicles. The membrane traffic of an epitope-tagged form of VAMP-2 (VAMP-TAg) was observed in transfected cells to identify sequence requirements for recycling of a synaptic vesicle membrane protein. In the neuroendocrine PC12 cell line VAMP-TAg is found not only in synaptic vesicles, but also in endosomes and on the plasma membrane. Endocytosis of VAMP-TAg is a rapid and saturable process. At high expression levels VAMP-TAg accumulates at the cell surface. Rapid endocytosis of VAMP-TAg also occurs in transfected CHO cells and is therefore independent of other synaptic proteins. The majority of the measured endocytosis is not directly into synaptic vesicles since mutations in VAMP-TAg that enhance synaptic vesicle targeting did not affect endocytosis. Nonetheless, mutations that inhibited synaptic vesicle targeting, in particular replacement of methionine-46 by alanine, inhibited endocytosis by 85% in PC12 cells and by 35% in CHO cells. These results demonstrate that the synaptic vesicle targeting signal is also used for endocytosis and can be recognized in cells lacking synaptic vesicles.