Extracellular immune responses to ascomycete and oomycete pathogens in Arabidopsis are dependent on vesicle- associated secretion mediated by the SNARE proteins PEN1 syntaxin, SNAP33 and endomembrane-resident VAMP721/722. Continuous movement of functional GFP-VAMP722 to and from the plasma membrane in non-stimulated cells reflects the second proposed function of VAMP721/722 in constitutive secretion during plant growth and development. Application of the bacterium-derived elicitor flg22 stabilizes VAMP721/722 that are otherwise constitutively degraded via the 26S proteasome pathway. Depletion of VAMP721/722 levels by reducing VAMP721/722 gene dosage enhances flg22-induced seedling growth inhibition in spite of elevated VAMP721/722 abundance. We therefore propose that plants prioritize the deployment of the corresponding secretory pathway for defense over plant growth. Interstingly, VAMP721/722 specifically interact in vitro and in vivo with the plasma membrane syntaxin SYP132 that is required for plant growth and resistance to bacteria. This suggests that the plant growth/immunity-involved VAMP721/722 form SNARE complexes with multiple plasma membrane syntaxins to discharge cue-dependent cargo molecules.
plant growth; plant immune responses; PM syntaxins; secretion; VAMP721/722
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.
The partitioning membrane of dividing plant cells is made by homotypic fusion of trans-Golgi network–derived membrane vesicles delivered to the division plane. The cytokinesis-specific syntaxin of Arabidopsis forms two different types of SNARE complexes, which can functionally replace each other in membrane fusion during cytokinesis.
Membrane fusion is mediated by soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes. Although membrane fusion is required for separating daughter cells in eukaryotic cytokinesis, the SNARE complexes involved are not known. In plants, membrane vesicles targeted to the cell division plane fuse with one another to form the partitioning membrane, progressing from the center to the periphery of the cell. In Arabidopsis, the cytokinesis-specific Qa-SNARE KNOLLE interacts with two other Q-SNAREs, SNAP33 and novel plant-specific SNARE 11 (NPSN11), whose roles in cytokinesis are not clear. Here we show by coimmunoprecipitation that KNOLLE forms two SNARE complexes that differ in composition. One complex is modeled on the trimeric plasma membrane type of SNARE complex and includes, in addition to KNOLLE, the promiscuous Qb,c-SNARE SNAP33 and the R-SNARE vesicle-associated membrane protein (VAMP) 721,722, also involved in innate immunity. In contrast, the other KNOLLE-containing complex is tetrameric and includes Qb-SNARE NPSN11, Qc-SNARE SYP71, and VAMP721,722. Elimination of only one or the other type of KNOLLE complex by mutation, including the double mutant npsn11
syp71, causes a mild or no cytokinesis defect. In contrast, the two double mutants snap33 npsn11 and snap33 syp71 eliminate both types of KNOLLE complexes and display knolle-like cytokinesis defects. Thus the two distinct types of KNOLLE complexes appear to jointly mediate membrane fusion in Arabidopsis cytokinesis.
The growing pollen tube apex is dedicated to balancing exo- and endocytic processes to form a rapidly extending tube. As perturbation of either tends to cause a morphological phenotype, this system provides tractable model for studying these processes. Vesicle-associated membrane protein 7s (VAMP7s) are members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family that mediate cognate membrane fusion but their role in pollen tube growth has not been investigated. This manuscript identifies PiVAMP726 of Petunia inflata as a pollen-specific VAMP7 that localizes to the inverted cone of transport vesicles at the pollen tube tip. The endocytic marker FM4-64 was found to colocalize with yellow fluorescent protein (YFP)-PiVAMP726, which is consistent with PiVAMP726 containing an amino-acid motif implicated in endosomal localization, At high overexpression levels, YFP- PiVAMP726 inhibited growth and caused the formation of novel membrane compartments within the pollen tube tip. Functional dissection of PiVAMP726 implicated the N-terminal longin domain in negative regulation of the SNARE activity, but not localization of PiVAMP726. Expression of the constitutively active C-terminal SNARE domain alone, in pollen tubes, generated similar phenotypes to the full-length protein, but the truncated domain was more potent than the wild-type protein at both inhibiting growth and forming the novel membrane compartments. Both endo- and exocytic markers localized to these compartments in addition to YFP-PiVAMP726, leading to the speculation that PiVAMP726 might be involved in the recycling of endocytic vesicles in tip growth.
Endocytosis; pollen tube growth; SNARE; VAMP7
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.
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.
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.
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
VARP is a Rab32/38 effector that also binds to the endosomal/lysosomal R-SNARE VAMP7. VARP binding regulates VAMP7 participation in SNARE complex formation and can therefore influence VAMP7-mediated membrane fusion events. Mutant versions of VARP that cannot bind Rab32:GTP, designed on the basis of the VARP ankyrin repeat/Rab32:GTP complex structure described here, unexpectedly retain endosomal localization, showing that VARP recruitment is not dependent on Rab32 binding. We show that recruitment of VARP to the endosomal membrane is mediated by its direct interaction with VPS29, a subunit of the retromer complex, which is involved in trafficking from endosomes to the TGN and the cell surface. Transport of GLUT1 from endosomes to the cell surface requires VARP, VPS29, and VAMP7 and depends on the direct interaction between VPS29 and VARP. Finally, we propose that endocytic cycling of VAMP7 depends on its interaction with VARP and, consequently, also on retromer.
•VARP recruitment to endosomes does not depend on its interaction with Rab32:GTP•VARP contains two Zinc-binding CHPLCxCxxC motifs that bind the retromer subunit VPS29•VARP is recruited to endosomes through its interaction with VPS29•VARP, retromer, and VAMP7 are all involved in trafficking of GLUT1 to the cell surface
The VAMP7-binding Rab32-effector and Rab21GEF VARP binds directly to the retromer subunit VPS29. VARP’s interaction with VPS29 recruits it on to endosomes, thereby linking the cargo sorting retromer complex with an R-SNARE involved in endosomal function. All three are shown to be involved in endosome to cell surface transport.
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.
Upon stimulation of insulin signalling or contraction-induced AMP-activated protein kinase (AMPK) activation, the glucose transporter GLUT4 and the long-chain fatty acid (LCFA) transporter CD36 similarly translocate from intracellular compartments to the plasma membrane of cardiomyocytes to increase uptake of glucose and LCFA, respectively. This similarity in regulation of GLUT4 traffic and CD36 traffic suggests that the same families of trafficking proteins, including vesicle-associated membrane proteins (VAMPs), are involved in both processes. While several VAMPs have been implicated in GLUT4 traffic, nothing is known about the putative function of VAMPs in CD36 traffic. Therefore, we compared the involvement of the myocardially produced VAMP isoforms in insulin- or contraction-induced GLUT4 and CD36 translocation.
Five VAMP isoforms were silenced in HL-1 cardiomyocytes. The cells were treated with insulin or the contraction-like AMPK activator oligomycin or were electrically stimulated to contract. Subsequently, GLUT4 and CD36 translocation as well as substrate uptake were measured.
Three VAMPs were demonstrated to be necessary for both GLUT4 and CD36 translocation, either specifically in insulin-treated cells (VAMP2, VAMP5) or in oligomycin/contraction-treated cells (VAMP3). In addition, there are VAMPs specifically involved in either GLUT4 traffic (VAMP7 mediates basal GLUT4 retention) or CD36 traffic (VAMP4 mediates insulin- and oligomycin/contraction-induced CD36 translocation).
The involvement of distinct VAMP isoforms in both GLUT4 and CD36 translocation indicates that CD36 translocation, just like GLUT4 translocation, is a vesicle-mediated process dependent on soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. The ability of other VAMPs to discriminate between GLUT4 and CD36 translocation allows the notion that myocardial substrate preference can be modulated by these VAMPs.
Cardiomyocytes; CD36; Contraction; GLUT4; Insulin; SNARE; VAMP
The trans-Golgi network (TGN) plays a pivotal role in directing proteins in the secretory pathway to the appropriate cellular destination. VAMP4, a recently discovered member of the vesicle-associated membrane protein (VAMP) family of trafficking proteins, has been suggested to play a role in mediating TGN trafficking. To better understand the function of VAMP4, we examined its precise subcellular distribution. Indirect immunofluorescence and electron microscopy revealed that the majority of VAMP4 localized to tubular and vesicular membranes of the TGN, which were in part coated with clathrin. In these compartments, VAMP4 was found to colocalize with the putative TGN-trafficking protein syntaxin 6. Additional labeling was also present on clathrin-coated and noncoated vesicles, on endosomes and the medial and trans side of the Golgi complex, as well as on immature secretory granules in PC12 cells. Immunoprecipitation of VAMP4 from rat brain detergent extracts revealed that VAMP4 exists in a complex containing syntaxin 6. Converging lines of evidence implicate a role for VAMP4 in TGN-to-endosome transport.
Insulin treatment of fat cells results in the translocation of the
insulin-responsive glucose transporter type 4, GLUT4, from
intracellular compartments to the plasma membrane. However, the precise
nature of these intracellular GLUT4-carrying compartments is debated.
To resolve the nature of these compartments, we have performed an
extensive morphological analysis of GLUT4-containing compartments,
using a novel immunocytochemical technique enabling high labeling
efficiency and 3-d resolution of cytoplasmic rims isolated
from rat epididymal adipocytes. In basal cells, GLUT4 was localized to
three morphologically distinct intracellular structures: small
vesicles, tubules, and vacuoles. In response to insulin the increase of
GLUT4 at the cell surface was compensated by a decrease in small
vesicles, whereas the amount in tubules and vacuoles was unchanged.
Under basal conditions, many small GLUT4 positive vesicles also
contained IRAP (88%) and the v-SNARE, VAMP2 (57%) but not markers of
sorting endosomes (EEA1), late endosomes, or lysosomes (lgp120). A
largely distinct population of GLUT4 vesicles (56%) contained the
cation-dependent mannose 6-phosphate receptor (CD-MPR), a marker
protein that shuttles between endosomes and the trans-Golgi network
(TGN). In response to insulin, GLUT4 was recruited both from VAMP2 and
CD-MPR positive vesicles. However, while the concentration of GLUT4 in
the remaining VAMP2-positive vesicles was unchanged, the concentration
of GLUT4 in CD-MPR-positive vesicles decreased. Taken together, we
provide morphological evidence indicating that, in response to insulin,
GLUT4 is recruited to the plasma membrane by fusion of preexisting
VAMP2-carrying vesicles as well as by sorting from the dynamic
The Golgi apparatus forms a twisted ribbon-like network in the juxtanuclear region of vertebrate cells. Vesicle-associated membrane protein 4 (VAMP4), a v-SNARE protein expressed exclusively in the vertebrate trans-Golgi network (TGN), plays a role in retrograde trafficking from the early endosome to the TGN, although its precise function within the Golgi apparatus remains unclear. To determine whether VAMP4 plays a functional role in maintaining the structure of the Golgi apparatus, we depleted VAMP4 gene expression using RNA interference technology. Depletion of VAMP4 from HeLa cells led to fragmentation of the Golgi ribbon. These fragments were not uniformly distributed throughout the cytoplasm, but remained in the juxtanuclear area. Electron microscopy and immunohistochemistry showed that in the absence of VAMP4, the length of the Golgi stack was shortened, but Golgi stacking was normal. Anterograde trafficking was not impaired in VAMP4-depleted cells, which contained intact microtubule arrays. Depletion of the cognate SNARE partners of VAMP4, syntaxin 6, syntaxin 16, and Vti1a also disrupted the Golgi ribbon structure. Our findings suggested that the maintenance of Golgi ribbon structure requires normal retrograde trafficking from the early endosome to the TGN, which is likely to be mediated by the formation of VAMP4-containing SNARE complexes.
VAMP4; SNARE; Golgi ribbon structure; Golgi fragmentation; RNAi; Membrane transport
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.
Epithelial cells elaborate specialized domains that have distinct protein and lipid compositions, including the apical and basolateral surfaces and primary cilia. Maintaining the identity of these domains is required for proper cell function, and requires the efficient and selective SNARE-mediated fusion of vesicles containing newly synthesized and recycling proteins with the proper target membrane. Multiple pathways exist to deliver newly synthesized proteins to the apical surface of kidney cells, and the post-Golgi SNAREs, or VAMPs, involved in these distinct pathways have not been identified. VAMP7 has been implicated in apical protein delivery in other cell types, and we hypothesized that this SNARE would have differential effects on the trafficking of apical proteins known to take distinct routes to the apical surface in kidney cells. VAMP7 expressed in polarized Madin Darby canine kidney cells colocalized primarily with LAMP2-positive compartments, and siRNA-mediated knockdown modulated lysosome size, consistent with the known function of VAMP7 in lysosomal delivery. Surprisingly, VAMP7 knockdown had no effect on apical delivery of numerous cargoes tested, but did decrease the length and frequency of primary cilia. Additionally, VAMP7 knockdown disrupted cystogenesis in cells grown in a three-dimensional basement membrane matrix. The effects of VAMP7 depletion on ciliogenesis and cystogenesis are not directly linked to the disruption of lysosomal function, as cilia lengths and cyst morphology were unaffected in an MDCK lysosomal storage disorder model. Together, our data suggest that VAMP7 plays an essential role in ciliogenesis and lumen formation. To our knowledge, this is the first study implicating an R-SNARE in ciliogenesis and cystogenesis.
Background: Exocytic delivery of the renal co-transporter NKCC2 to the cell surface is a major mechanism for NaCl reabsorption.
Results: We describe a mechanism that mediates cAMP-stimulated NKCC2 delivery in renal cells.
Conclusion: Vesicle fusion protein VAMP2 interacts with NKCC2 and mediates cAMP-stimulated NKCC2 exocytic delivery.
Significance: The molecular mechanism mediating NKCC2 exocytic delivery could provide new targets for treatment of hypertension.
In the kidney, epithelial cells of the thick ascending limb (TAL) reabsorb NaCl via the apical Na+/K+/2Cl− co-transporter NKCC2. Steady-state surface NKCC2 levels in the apical membrane are maintained by a balance between exocytic delivery, endocytosis, and recycling. cAMP is the second messenger of hormones that enhance NaCl absorption. cAMP stimulates NKCC2 exocytic delivery via protein kinase A (PKA), increasing steady-state surface NKCC2. However, the molecular mechanism involved has not been studied. We found that several members of the SNARE family of membrane fusion proteins are expressed in TALs. Here we report that NKCC2 co-immunoprecipitates with VAMP2 in rat TALs, and they co-localize in discrete domains at the apical surface. cAMP stimulation enhanced VAMP2 exocytic delivery to the plasma membrane of renal cells, and stimulation of PKA enhanced VAMP2-NKCC2 co-immunoprecipitation in TALs. In vivo silencing of VAMP2 but not VAMP3 in TALs blunted cAMP-stimulated steady-state surface NKCC2 expression and completely blocked cAMP-stimulated NKCC2 exocytic delivery. VAMP2 was not involved in constitutive NKCC2 delivery. We concluded that VAMP2 but not VAMP3 selectively mediates cAMP-stimulated NKCC2 exocytic delivery and surface expression in TALs. We also demonstrated that cAMP stimulation enhances VAMP2 exocytosis and promotes VAMP2 interaction with NKCC2.
Cyclic AMP (cAMP); Epithelial Cell; Exocytosis; Kidney; Membrane Trafficking; SNARE Proteins; Na-K-Cl Co-transporter 2 (NKCC2); Epithelial Sodium Transport
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
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
Intracellular membrane traffic is an essential component of the membrane remodeling that supports lamellipodium extension during cell adhesion. The membrane trafficking pathways that contribute to cell adhesion have not been fully elucidated, but recent studies have implicated SNARE proteins. Here, the functions of several SNAREs (SNAP23, VAMP3, VAMP4 and syntaxin13) are characterized during the processes of cell spreading and membrane ruffling.
We report the first description of a SNARE complex, containing SNAP23, syntaxin13 and cellubrevin/VAMP3, that is induced by cell adhesion to an extracellular matrix. Impairing the function of the SNAREs in the complex using inhibitory SNARE domains disrupted the recycling endosome, impeded delivery of integrins to the cell surface, and reduced haptotactic cell migration and spreading. Blocking SNAP23 also inhibited the formation of PMA-stimulated, F-actin-rich membrane ruffles; however, membrane ruffle formation was not significantly altered by inhibition of VAMP3 or syntaxin13. In contrast, membrane ruffling, and not cell spreading, was sensitive to inhibition of two SNAREs within the biosynthetic secretory pathway, GS15 and VAMP4. Consistent with this, formation of a complex containing VAMP4 and SNAP23 was enhanced by treatment of cells with PMA. The results reveal a requirement for the function of a SNAP23-syntaxin13-VAMP3 complex in the formation of lamellipodia during cell adhesion and of a VAMP4-SNAP23-containing complex during PMA-induced membrane ruffling.
Our findings suggest that different SNARE-mediated trafficking pathways support membrane remodeling during ECM-induced lamellipodium extension and PMA-induced ruffle formation, pointing to important mechanistic differences between these processes.
Vasopressin regulates the fusion of the water channel aquaporin 2 (AQP2) to the apical membrane of the renal collecting-duct principal cells and several lines of evidence indicate that SNARE proteins mediate this process. In this work MCD4 renal cells were used to investigate the functional role of a set of Q- and R-SNAREs, together with that of Munc18b as a negative regulator of the formation of the SNARE complex. Both VAMP2 and VAMP3 were associated with immunoisolated AQP2 vesicles, whereas syntaxin 3 (Stx3), SNAP23 and Munc18 were associated with the apical plasma membrane. Co-immunoprecipitation experiments indicated that Stx3 forms complexes with VAMP2, VAMP3, SNAP23 and Munc18b. Protein knockdown coupled to apical surface biotinylation demonstrated that reduced levels of the R-SNAREs VAMP2 and VAMP3, and the Q-SNAREs Stx3 and SNAP23 strongly inhibited AQP2 fusion at the apical membrane. In addition, knockdown of Munc18b promoted a sevenfold increase of AQP2 fused at the plasma membrane without forskolin stimulation.
Taken together these findings propose VAMP2, VAMP3, Stx3 and SNAP23 as the complementary set of SNAREs responsible for AQP2-vesicle fusion into the apical membrane, and Munc18b as a negative regulator of SNARE-complex formation in renal collecting-duct principal cells.
Aquaporin2; Vasopressin; VAMP; SNARE; Syntaxin; Munc18; Exocytosis
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.
Body water balance is controlled by vasopressin, which regulates Aquaporin-2 (AQP2) water channels in kidney collecting duct cells by vesicular trafficking between intracellular vesicles and the plasma membrane. To examine the molecular apparatus involved in vesicle trafficking and vasopressin regulation of AQP2 in collecting duct cells, we tested if targeting proteins expressed in the synaptic vesicles, namely vesicle-associated membrane proteins 1 and 2 (VAMP1 and 2), are expressed in kidney collecting duct. Immunoblotting revealed specific labeling of VAMP2 (18-kD band) but not VAMP1 in membrane fractions prepared from kidney inner medulla. Controls using preadsorbed antibody or preimmune serum were negative. Bands of identical molecular size were detected in immunoblots of brain membrane vesicles and purified synaptic vesicles. VAMP2 in kidney membranes was cleaved by tetanus toxin, revealing a tetanus toxin-sensitive VAMP homologue. Similarly, tetanus toxin cleaved VAMP2 in synaptic vesicles. In kidney inner medulla, VAMP2 was predominantly expressed in the membrane fraction enriched for intracellular vesicles, with little or no VAMP2 in the plasma membrane enriched fraction. This was confirmed by immunocytochemistry using semithin cryosections, which showed mainly vesicular labeling in collecting duct principal cells, with no labeling of intercalated cells. VAMP2 immunolabeling colocalized with AQP2 labeling in intracellular vesicles, as determined by immunoelectron microscopy after double immunolabeling of isolated vesicles. Quantitative analysis of 1,310 vesicles revealed a highly significant association of both AQP2 and VAMP2 in the same vesicles (P < 0.0001). Furthermore, the presence of AQP2 in vesicles immunoisolated with anti-VAMP2 antibodies was confirmed by immunoblotting. In conclusion, VAMP2, a component of the neuronal SNARE complex, is expressed in vesicles carrying AQP2, suggesting a role in vasopressin-regulated vesicle trafficking of AQP2 water channels.
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.
Insulin stimulates glucose transport in adipocytes by translocation of the glucose transporter (GLUT-4) from an intracellular site to the cell surface. We have characterized different synaptobrevin/vesicle- associated membrane protein (VAMP) homologues in adipocytes and studied their intracellular distribution with respect to GLUT-4. VAMP-1, VAMP- 2, and cellubrevin cDNAs were isolated from a 3T3-L1 adipocyte expression library. VAMP-2 and cellubrevin were: (a) the most abundant isoforms in adipocytes, (b) detectable in all insulin responsive tissues, (c) translocated to the cell surface in response to insulin, and (d) found in immunoadsorbed GLUT-4 vesicles. To further define their intracellular distribution, 3T3-L1 adipocytes were incubated with a transferrin/HRP conjugate (Tf/HRP) and endosomes ablated following addition of DAB and H2O2. While this resulted in ablation of > 90% of the transferrin receptor (TfR) and cellubrevin found in intracellular membranes, 60% of GLUT-4 and 90% of VAMP-2 was not ablated. Immuno-EM on intracellular vesicles from adipocytes revealed that VAMP-2 was colocalized with GLUT-4, whereas only partial colocalization was observed between GLUT-4 and cellubrevin. These studies show that two different v-SNAREs, cellubrevin and VAMP-2, are partially segregated in different intracellular compartments in adipocytes, implying that they may define separate classes of secretory vesicles in these cells. We conclude that a proportion of GLUT-4 is found in recycling endosomes in nonstimulated adipocytes together with cellubrevin and the transferrin receptor. In addition, GLUT-4 and VAMP-2 are selectively enriched in a postendocytic compartment. Further study is required to elucidate the function of this latter compartment in insulin-responsive cells.