Synaptotagmin IV is necessary for the maturation of secretory granules in PC12 cells

In neuroendocrine PC12 cells, immature secretory granules (ISGs) mature through homotypic fusion and membrane remodeling. We present evidence that the ISG-localized synaptotagmin IV (Syt IV) is involved in ISG maturation. Using an in vitro homotypic fusion assay, we show that the cytoplasmic domain (CD) of Syt IV, but not of Syt I, VII, or IX, inhibits ISG homotypic fusion. Moreover, Syt IV CD binds specifically to ISGs and not to mature secretory granules (MSGs), and Syt IV binds to syntaxin 6, a SNARE protein that is involved in ISG maturation. ISG homotypic fusion was inhibited in vivo by small interfering RNA-mediated depletion of Syt IV. Furthermore, the Syt IV CD, as well as Syt IV depletion, reduces secretogranin II (SgII) processing by prohormone convertase 2 (PC2). PC2 is found mostly in the proform, suggesting that activation of PC2 is also inhibited. Granule formation, and the sorting of SgII and PC2 from the trans-Golgi network into ISGs and MSGs, however, is not affected. We conclude that Syt IV is an essential component for secretory granule maturation.     

Homotypic Fusion of Immature Secretory Granules during Maturation in a Cell-free Assay

The biogenesis of secretory granules embodies several morphological and biochemical changes. In particular, in neuroendocrine cells maturation of secretory granules is characterized by an increase in size which has been proposed to reflect homotypic fusion of immature secretory granules (ISGs). Here we describe an assay that provides the first biochemical evidence for such a fusion event and allows us to analyze its regulation. The assay reconstitutes homotypic fusion between one population of ISGs containing a [³⁵S]sulfate-labeled substrate, secretogranin II (SgII), and a second population containing the prohormone convertase PC2. Both substrate and enzyme are targeted exclusively to ISGs. Fusion is measured by quantification of a cleavage product of SgII produced by PC2. With this assay we show that fusion only occurs between ISGs and not between ISGs and MSGs, is temperature dependent, and requires ATP and GTP and cytosolic proteins. NSF (N-ethylmaleimide–sensitive fusion protein) is amongst the cytosolic proteins required, whereas we could not detect a requirement for p97. The ability to reconstitute ISG fusion in a cell-free assay is an important advance towards the identification of molecules involved in the maturation of secretory granules and will increase our understanding of this process.     

Hrs regulates early endosome fusion by inhibiting formation of an endosomal SNARE complex

Movement through the endocytic pathway occurs principally via a series of membrane fusion and fission reactions that allow sorting of molecules to be recycled from those to be degraded. Endosome fusion is dependent on SNARE proteins, although the nature of the proteins involved and their regulation has not been fully elucidated. We found that the endosome-associated hepatocyte responsive serum phosphoprotein (Hrs) inhibited the homotypic fusion of early endosomes. A region of Hrs predicted to form a coiled coil required for binding the Q-SNARE, SNAP-25, mimicked the inhibition of endosome fusion produced by full-length Hrs, and was sufficient for endosome binding. SNAP-25, syntaxin 13, and VAMP2 were bound from rat brain membranes to the Hrs coiled-coil domain. Syntaxin 13 inhibited early endosomal fusion and botulinum toxin/E inhibition of early endosomal fusion was reversed by addition of SNAP-25(150-206), confirming a role for syntaxin 13, and establishing a role for SNAP-25 in endosomal fusion. Hrs inhibited formation of the syntaxin 13-SNAP-25-VAMP2 complex by displacing VAMP2 from the complex. These data suggest that SNAP-25 is a receptor for Hrs on early endosomal membranes and that the binding of Hrs to SNAP-25 on endosomal membranes inhibits formation of a SNARE complex required for homotypic endosome fusion.     

3-Methylcholanthrene elicits DNA adduct formation in the CYP1A1 promoter region and attenuates reporter gene expression in rat H4IIE cells

Since it was reported that components of immature secretory granules (ISGs) are different from those of mature secretory granules (MSGs) in rat parotid acinar cells, we have been considering that components of secretory granules (SGs) change dynamically during granule maturation. As the first step to understand the mechanism of granule maturation, we separated low-density detergent-resistant membrane fractions (DRMs) from purified SGs of rat parotid gland. When SGs were lysed by the detergent Brij-58, syntaxin6 and VAMP4 were found in DRMs that were different from the GM1a-rich DRMs containing VAMP2. Because syntaxin6 and VAMP4 are known to be related to granule formation, we attempted to separate DRMs from ISGs. To enrich for ISGs, glands were removed from rats 5h after intraperitoneal injection of isoproterenol and used to purify the newly synthesized granules. Compared to mature granules prepared without injection, these newly formed granules were lower in density and contained higher concentrations of syntaxin6, VAMP4, and gamma-adaptin. This composition is consistent with the characterizations of ISGs. DRMs isolated from the newly formed granules were GM1a-rich and contained syntaxin6, VAMP4, and VAMP2 together. Thus, our findings suggest that syntaxin6 and VAMP4 associate with a GM1a-rich membrane microdomain during granule formation but enter a separate membrane microdomain before transport from granules during maturation.     

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

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 alpha-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.     

Docking of liposomes to planar surfaces mediated by trans-SNARE complexes

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a key role in membrane fusion in the secretory pathway. In vitro, SNAREs spontaneously assemble into helical SNARE complexes with the transmembrane domains at the C-terminal end. During fusion, SNAREs are thought to bridge the two membranes and assemble in a zipper-like fashion, pulling the membranes together and initiating fusion. However, it is not clear to what extent SNARE assembly contributes to membrane attachment and membrane fusion. Using the neuronal SNAREs synaptobrevin (VAMP), SNAP-25, and syntaxin as examples, we show here that liposomes containing synaptobrevin firmly attach to planar surfaces containing immobilized syntaxin. Attachment requires the formation of SNARE complexes because it is dependent on the presence of SNAP-25. Binding is competed for by soluble SNARE fragments, with noncognate SNAREs such as endobrevin (VAMP8), VAMP4, and VAMP7 (Ti-VAMP) being effective but less potent in some cases. Furthermore, although SNAP-23 is unable to substitute for SNAP-25 in the attachment assay, it forms complexes of comparable stability and is capable of substituting in liposome fusion assays. Vesicle attachment is initiated by SNARE assembly at the N-terminal end of the helix bundle. We conclude that SNAREs can indeed form stable trans-complexes that result in vesicle attachment if progression to fusion is prevented, further supporting the zipper model of SNARE function.     

Vesicle-associated membrane protein 8 (VAMP8) is a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) selectively required for sequential granule-to-granule fusion

Compound exocytosis is found in many cell types and is the major form of regulated secretion in acinar and mast cells. Its key characteristic is the homotypic fusion of secretory granules. These then secrete their combined output through a single fusion pore to the outside. The control of compound exocytosis remains poorly understood. Although soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) such as syntaxin 2, SNAP23 (synaptosome-associated protein of 23 kDa), and SNAP25 have been suggested to play a role, none has been proven. Vesicle-associated membrane protein 8 (VAMP8) is a SNARE first associated with endocytic processes but more recently has been suggested as an R-SNARE in regulated exocytosis. Secretion in acinar cells is reduced when VAMP8 function is inhibited and is less in VAMP8 knock-out mice. Based on electron microscopy experiments, it was suggested that VAMP8 may be involved in compound exocytosis. Here we have tested the hypothesis that VAMP8 controls homotypic granule-to-granule fusion during sequential compound exocytosis. We use a new assay to distinguish primary fusion events (fusion with the cell membrane) from secondary fusion events (granule-granule fusion). Our data show the pancreatic acinar cells from VAMP8 knock-out animals have a specific reduction in secondary granule fusion but that primary granule fusion is unaffected. Furthermore, immunoprecipitation experiments show syntaxin 2 association with VAMP2, whereas syntaxin 3 associates with VAMP8. Taken together our data indicate that granule-to-granule fusion is regulated by VAMP8 containing SNARE complexes distinct from those that regulate primary granule fusion.     

Ternary SNARE complexes are enriched in lipid rafts during mast cell exocytosis

Lipid rafts are membrane microdomains rich in cholesterol and glycosphingolipids that have been implicated in the regulation of intracellular protein trafficking. During exocytosis, a class of proteins termed SNAREs mediate secretory granule-plasma membrane fusion. To investigate the role of lipid rafts in secretory granule exocytosis, we examined the raft association of SNARE proteins and SNARE complexes in rat basophilic leukemia (RBL) mast cells. The SNARE protein SNAP-23 co-localized with a lipid raft marker and was present in detergent-insoluble lipid raft microdomains in RBL cells. By contrast, only small amounts (<20%) of the plasma membrane SNARE syntaxin 4 or the granule-associated SNARE vesicle-associated membrane protein (VAMP)-2 were present in these microdomains. Despite this, essentially all syntaxin 4 and most of VAMP-2 in these rafts were present in SNARE complexes containing SNAP-23, while essentially none of these complexes were present in nonraft membranes. Whereas SNAP-23 is membrane anchored by palmitoylation, the association of the transmembrane protein syntaxin 4 with lipid rafts was because of its binding to SNAP-23. After stimulating mast cells exocytosis, the amount of syntaxin 4 and VAMP-2 present in rafts increased twofold, and these proteins were now present in raft-associated phospho-SNAP-23/syntaxin 4/VAMP-2 complexes, revealing differential association of SNARE fusion complexes during the process of regulated exocytosis.     

Soluble NSF attachment protein receptors (SNAREs) in RBL-2H3 mast cells: functional role of syntaxin 4 in exocytosis and identification of a vesicle-associated membrane protein 8-containing secretory compartment

Mast cells upon stimulation through high affinity IgE receptors massively release inflammatory mediators by the fusion of specialized secretory granules (related to lysosomes) with the plasma membrane. Using the RBL-2H3 rat mast cell line, we investigated whether granule secretion involves components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery. Several isoforms of each family of SNARE proteins were expressed. Among those, synaptosome-associated protein of 23 kDa (SNAP23) was central in SNARE complex formation. Within the syntaxin family, syntaxin 4 interacted with SNAP23 and all vesicle-associated membrane proteins (VAMPs) examined, except tetanus neurotoxin insensitive VAMP (TI-VAMP). Overexpression of syntaxin 4, but not of syntaxin 2 nor syntaxin 3, caused inhibition of FcepsilonRI-dependent exocytosis. Four VAMP proteins, i.e., VAMP2, cellubrevin, TI-VAMP, and VAMP8, were present on intracellular membrane structures, with VAMP8 residing mainly on mediator-containing secretory granules. We suggest that syntaxin 4, SNAP23, and VAMP8 may be involved in regulation of mast cell exocytosis. Furthermore, these results are the first demonstration that the nonneuronal VAMP8 isoform, originally localized on early endosomes, is present in a regulated secretory compartment.     

Secretory granule biogenesis: rafting to the SNARE

Regulated secretion of hormones occurs when a cell receives an external stimulus, triggering the secretory granules to undergo fusion with the plasma membrane and release their content into the extracellular milieu. The formation of a mature secretory granule (MSG) involves a series of discrete and unique events such as protein sorting, formation of immature secretory granules (ISGs), prohormone processing and vesicle fusion. Regulated secretory proteins (RSPs), the proteins stored and secreted from MSGs, contain signals or domains to direct them into the regulated secretory pathway. Recent data on the role of specific domains in RSPs involved in sorting and aggregation suggest that the cell-type-specific composition of RSPs in the trans-Golgi network (TGN) has an important role in determining how the RSPs get into ISGs. The realization that lipid rafts are implicated in sorting RSPs in the TGN and the identification of SNARE molecules represent further major advances in our understanding of how MSGs are formed. At the heart of these findings is the elucidation of molecular mechanisms driving protein--lipid and protein--protein interactions specific for secretory granule biogenesis.     

Combinatorial SNARE complexes modulate the secretion of cytoplasmic granules in human neutrophils

Mobilization of human neutrophil granules is critical for the innate immune response against infection and for the outburst of inflammation. Human neutrophil-specific and tertiary granules are readily exocytosed upon cell activation, whereas azurophilic granules are mainly mobilized to the phagosome. These cytoplasmic granules appear to be under differential secretory control. In this study, we show that combinatorial soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes with vesicle-associated membrane proteins (VAMPs), 23-kDa synaptosome-associated protein (SNAP-23), and syntaxin 4 underlie the differential mobilization of granules in human neutrophils. Specific and tertiary granules contained VAMP-1, VAMP-2, and SNAP-23, whereas the azurophilic granule membranes were enriched in VAMP-1 and VAMP-7. Ultrastructural, coimmunoprecipitation, and functional assays showed that SNARE complexes containing VAMP-1, VAMP-2, and SNAP-23 mediated the rapid exocytosis of specific/tertiary granules, whereas VAMP-1 and VAMP-7 mainly regulated the secretion of azurophilic granules. Plasma membrane syntaxin 4 acted as a general target SNARE for the secretion of the distinct granule populations. These data indicate that at least two SNARE complexes, made up of syntaxin 4/SNAP-23/VAMP-1 and syntaxin 4/SNAP-23/VAMP-2, are involved in the exocytosis of specific and tertiary granules, whereas interactions between syntaxin 4 and VAMP-1/VAMP-7 are involved in the exocytosis of azurophilic granules. Our data indicate that quantitative and qualitative differences in SNARE complex formation lead to the differential mobilization of the distinct cytoplasmic granules in human neutrophils, and a higher capability to form diverse SNARE complexes renders specific/tertiary granules prone to exocytosis.     

A novel site of action for alpha-SNAP in the SNARE conformational cycle controlling membrane fusion

Regulated exocytosis in neurons and neuroendocrine cells requires the formation of a stable soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of synaptobrevin-2/vesicle-associated membrane protein 2, synaptosome-associated protein of 25 kDa (SNAP-25), and syntaxin 1. This complex is subsequently disassembled by the concerted action of alpha-SNAP and the ATPases associated with different cellular activities-ATPase N-ethylmaleimide-sensitive factor (NSF). We report that NSF inhibition causes accumulation of alpha-SNAP in clusters on plasma membranes. Clustering is mediated by the binding of alpha-SNAP to uncomplexed syntaxin, because cleavage of syntaxin with botulinum neurotoxin C1 or competition by using antibodies against syntaxin SNARE motif abolishes clustering. Binding of alpha-SNAP potently inhibits Ca(2+)-dependent exocytosis of secretory granules and SNARE-mediated liposome fusion. Membrane clustering and inhibition of both exocytosis and liposome fusion are counteracted by NSF but not when an alpha-SNAP mutant defective in NSF activation is used. We conclude that alpha-SNAP inhibits exocytosis by binding to the syntaxin SNARE motif and in turn prevents SNARE assembly, revealing an unexpected site of action for alpha-SNAP in the SNARE cycle that drives exocytotic membrane fusion.     

Differential Effects of Munc18s on Multiple Degranulation-Relevant Trans-SNARE Complexes

Mast cell exocytosis, which includes compound degranulation and vesicle-associated piecemeal degranulation, requires multiple Q- and R- SNAREs. It is not clear how these SNAREs pair to form functional trans-SNARE complexes and how these trans-SNARE complexes are selectively regulated for fusion. Here we undertake a comprehensive examination of the capacity of two Q-SNARE subcomplexes (syntaxin3/SNAP-23 and syntaxin4/SNAP-23) to form fusogenic trans-SNARE complexes with each of the four granule-borne R-SNAREs (VAMP2, 3, 7, 8). We report the identification of at least six distinct trans-SNARE complexes under enhanced tethering conditions: i) VAMP2/syntaxin3/SNAP-23, ii) VAMP2/syntaxin4/SNAP-23, iii) VAMP3/syntaxin3/SNAP-23, iv) VAMP3/syntaxin4/SNAP-23, v) VAMP8/syntaxin3/SNAP-23, and vi) VAMP8/syntaxin4/SNAP-23. We show for the first time that Munc18a operates synergistically with SNAP-23-based non-neuronal SNARE complexes (i to iv) in lipid mixing, in contrast to Munc18b and c, which exhibit no positive effect on any SNARE combination tested. Pre-incubation with Munc18a renders the SNARE-dependent fusion reactions insensitive to the otherwise inhibitory R-SNARE cytoplasmic domains, suggesting a protective role of Munc18a for its cognate SNAREs. Our findings substantiate the recently discovered but unexpected requirement for Munc18a in mast cell exocytosis, and implicate post-translational modifications in Munc18b/c activation.     

nSec1 binds a closed conformation of syntaxin1A

The Sec1 family of proteins is proposed to function in vesicle trafficking by forming complexes with target membrane SNAREs (soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein [SNAP] receptors) of the syntaxin family. Here, we demonstrate, by using in vitro binding assays, nondenaturing gel electrophoresis, and specific neurotoxin treatment, that the interaction of syntaxin1A with the core SNARE components, SNAP-25 (synaptosome-associated protein of 25 kD) and VAMP2 (vesicle-associated membrane protein 2), precludes the interaction with nSec1 (also called Munc18 and rbSec1). Inversely, association of nSec1 and syntaxin1A prevents assembly of the ternary SNARE complex. Furthermore, using chemical cross-linking of rat brain membranes, we identified nSec1 complexes containing syntaxin1A, but not SNAP-25 or VAMP2. These results support the hypothesis that Sec1 proteins function as syntaxin chaperons during vesicle docking, priming, and membrane fusion.     

Formation of the full SNARE complex eliminates interactions of its individual protein components with the Kv2.1 channel

Previously, we have demonstrated physical and functional interactions of the voltage-gated potassium channel Kv2.1 with the plasma membrane protein components of the exocytotic SNARE complex, syntaxin 1A, and the t-SNARE, syntaxin 1A/SNAP-25, complex. Importantly, the physical interaction of Kv2.1 with syntaxin was shown to be involved in the facilitation of secretion from PC12 cells, which was independent of potassium currents. Recently, we showed that also VAMP2, the vesicular SNARE, interacts physically and functionally with Kv2.1. Here, we first set out to test the interaction of the full SNARE, syntaxin/SNAP-25/VAMP2, complex with the channel. Using the interaction of VAMP2 with Kv2.1 in Xenopus oocytes as a probe, we showed that coexpression of the t-SNARE complex with VAMP2 abolished the VAMP2 effect on channel inactivation and reduced the amount of VAMP2 that coprecipitated with Kv2.1. Further, in vitro pull down assays showed that the full SNARE complex failed to interact with Kv2.1 N- and C-termini in tandem, in contrast to the individual SNARE components. This suggests that the interactions of the SNARE components with Kv2.1 are abolished upon their recruitment into a full SNARE complex, which does not interact with the channel. Other important findings arising from the in vitro study are that the t-SNARE complex, in addition to syntaxin, interacts with a specific C-terminal channel domain, C1a, shown to mediate the facilitation of release by Kv2.1 and that the presence of Kv2.1 N-terminus has crucial contribution to these interactions. These findings provide important insights into the understanding of the complex molecular events involved in the novel phenomenon of secretion facilitation in neuroendocrine cells by Kv2.1.     

The last exon of SNAP-23 regulates granule exocytosis from mast cells

SNAP-25 and its ubiquitous homolog SNAP-23 are members of the SNARE family of proteins that regulate membrane fusion during exocytosis. Although SNAP-23 has been shown to participate in a variety of intracellular transport processes, the structural domains of SNAP-23 that are required for its interaction with other SNAREs have not been determined. By employing deletion mutagenesis we found that deletion of the amino-terminal 18 amino acids of SNAP-23 (encoded in the first exon) dramatically inhibited binding of SNAP-23 to both the target SNARE syntaxin and the vesicle SNARE vesicle-associated membrane protein(VAMP). By contrast, deletion of the carboxyl-terminal 23 amino acids (encoded in the last exon) of SNAP-23 does not affect SNAP-23 binding to syntaxin but profoundly inhibits its binding to VAMP. To determine the functional relevance of the modular structure of SNAP-23, we overexpressed SNAP-23 in cells possessing the capacity to undergo regulated exocytosis. Expression of human SNAP-23 in a rat mast cell line significantly enhanced exocytosis, and this effect was not observed in transfectants expressing the carboxyl-terminal VAMP-binding mutant of SNAP-23. Despite considerable amino acid identity, we found that human SNAP-23 bound to SNAREs more efficiently than did rat SNAP-23. These data demonstrate that the introduction of a "better" SNARE binder into secretory cells augments exocytosis and defines the carboxyl terminus of SNAP-23 as an essential regulator of exocytosis in mast cells.     

Amisyn,a novel syntaxin-binding protein that may regulate SNARE complex assembly

The regulation of SNARE complex assembly likely plays an important role in governing the specificity as well as the timing of membrane fusion. Here we identify a novel brain-enriched protein, amisyn, with a tomosyn- and VAMP-like coiled-coil-forming domain that binds specifically to syntaxin 1a and syntaxin 4 both in vitro and in vivo, as assessed by co-immunoprecipitation from rat brain. Amisyn is mostly cytosolic, but a fraction co-sediments with membranes. The amisyn coil domain can form SNARE complexes of greater thermostability than can VAMP2 with syntaxin 1a and SNAP-25 in vitro, but it lacks a transmembrane anchor and so cannot act as a v-SNARE in this complex. The amisyn coil domain prevents the SNAP-25 C-terminally mediated rescue of botulinum neurotoxin E inhibition of norepinephrine exocytosis in permeabilized PC12 cells to a greater extent than it prevents the regular exocytosis of these vesicles. We propose that amisyn forms nonfusogenic complexes with syntaxin 1a and SNAP-25, holding them in a conformation ready for VAMP2 to replace it to mediate the membrane fusion event, thereby contributing to the regulation of SNARE complex formation.     

Targeting of SNAP-25 to membranes is mediated by its association with the target SNARE syntaxin

The docking and fusion of synaptic vesicles with the presynaptic plasma membrane require the interaction of the vesicle-associated membrane protein VAMP with the plasma membrane proteins syntaxin and SNAP-25. Both of these proteins behave as integral membrane proteins, although they are unusual in that they insert into membranes post-translationally. Whereas VAMP and syntaxin possess hydrophobic transmembrane domains, SNAP-25 does not, and it is widely believed that SNAP-25 traffics to and inserts into membranes by post-translational palmitoylation. In pulse-chase biosynthesis studies, we now show that SNAP-25 and syntaxin rapidly bind to each other while still in the cytosol of neuroendocrine and transfected heterologous cells. Cell fractionation studies revealed that cytosolic SNAP-25.syntaxin complexes then traffic to and insert into membranes. Furthermore, the association of SNAP-25 with membranes is dramatically enhanced by syntaxin, and the transmembrane domain of syntaxin is essential for this effect. Surprisingly, despite the importance of the SNAP-25 palmitoylation domain for membrane anchoring at steady state, removal of this domain did not inhibit the initial association of newly synthesized SNAP-25 with membranes in the presence of syntaxin. These data demonstrate that the initial attachment of newly synthesized SNAP-25 to membranes is a consequence of its association with syntaxin and that it is only after syntaxin-mediated membrane tethering that SNAP-25 is palmitoylated.     

VAMP8/Endobrevin is a critical factor for the homotypic granule growth in pancreatic acinar cells

The delivery of newly-formed secretory content to the granule inventory occurs through direct fusion of recently formed granules and mature granules. The introduction of knockout mice allowed us to investigate the characteristics of the delivery process and to determine the core protein machinery required for granule growth. The SNARE machinery mediates membrane fusion and is essential for the granule lifecycle. In the current work, we use VAMP8 knockout mice to show that the SNARE machinery plays a critical role in the process of granule homotypic fusion. Consistent with this, the mutated mouse pancreatic acinar secretory granules are significantly smaller compared to the control group, demonstrating few granule profiles that might be the result of homotypic fusion.     

SNARE interactions are not selective. Implications for membrane fusion specificity

The SNARE hypothesis proposes that membrane trafficking specificity is mediated by preferential high affinity interactions between particular v (vesicle membrane)- and t (target membrane)-SNARE combinations. The specificity of interactions among a diverse set of SNAREs, however, is unknown. We have tested the SNARE hypothesis by analyzing potential SNARE complexes between five proteins of the vesicle-associated membrane protein (VAMP) family, three members of the synaptosome-associated protein-25 (SNAP-25) family and three members of the syntaxin family. All of the 21 combinations of SNAREs tested formed stable complexes. Sixteen were resistant to SDS denaturation, and most complexes thermally denatured between 70 and 90 degreesC. These results suggest that the specificity of membrane fusion is not encoded by the interactions between SNAREs.