Evidence for a role of the exocyst in insulin-stimulated Glut4 trafficking in 3T3-L1 adipocytes

Insulin stimulates glucose transport in adipocytes and muscle by inducing the redistribution of Glut4 from intracellular locations to the plasma membrane. The fusion of Glut4-containing vesicles at the plasma membrane is known to involve the target SNAREs syntaxin 4 and SNAP-23 and the vesicle SNARE VAMP2. Little is known about the initial docking of Glut4 vesicles with the plasma membrane. A recent report has implicated Exo70, a component of the mammalian exocyst complex, in the initial interaction of Glut4 vesicles with the adipocyte plasma membrane. Here, we have examined the role of two other exocyst components, rsec6 and rsec8. We show that insulin promotes a redistribution of rsec6 and rsec8 to the plasma membrane and to cytoskeletal fractions within 3T3-L1 adipocytes but does not modulate levels of these proteins co-localized with Glut4. We further show that adenoviral-mediated overexpression of either rsec6 or rsec8 increases the magnitude of insulin-stimulated glucose transport in 3T3-L1 adipocytes. By contrast, overexpression of rsec6 or rsec8 did not increase the extent of the secretion of adipsin or ACRP30 from adipocytes and had no discernible effect on transferrin receptor traffic. Collectively, our data support a role for the exocyst in insulin-stimulated glucose transport and suggest a model by which insulin-dependent relocation of the exocyst to the plasma membrane may contribute to the specificity of Glut4 vesicle docking and fusion with the adipocyte plasma membrane.     

The vesicle- and target-SNARE proteins that mediate Glut4 vesicle fusion are localized in detergent-insoluble lipid rafts present on distinct intracellular membranes

Insulin stimulates the fusion of intracellular vesicles containing the glucose transporter Glut4 with the plasma membrane in adipocytes and muscle cells. Glut4 vesicle fusion is thought to be catalyzed by the interaction of the vesicle soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptor VAMP2 with the target soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors SNAP-23 and syntaxin 4. Here, we use combined membrane fractionation, detergent solubility, and sucrose gradient flotation to demonstrate that the large majority (>70%) of SNAP-23 and a significant proportion of syntaxin 4 ( approximately 35%) are associated with plasma membrane lipid rafts in 3T3-L1 adipocytes. Furthermore, VAMP2 is shown to be concentrated in lipid rafts isolated from intracellular membranes. Insulin stimulation had no effect on the plasma membrane raft association of SNAP-23 or syntaxin 4 but promoted VAMP2 insertion into plasma membrane rafts. Immunofluorescence analysis revealed that SNAP-23 was clustered at the plasma membrane and almost completely segregated from the transferrin receptor. SNAP-23 distribution seemed to be distinct from caveolin-1, and clusters of SNAP-23 were dispersed after cholesterol extraction with methyl-beta-cyclodextrin, suggesting that the majority of SNAP-23 is associated with non-caveolar, cholesterol-rich lipid rafts. The results described implicate lipid rafts as important platforms for Glut4 vesicle fusion and suggest the hypothesis that such rafts may represent a spatial integration point of insulin signaling and membrane traffic.     

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.     

Syntaxin 4 in 3T3-L1 adipocytes: regulation by insulin and participation in insulin-dependent glucose transport.

Syntaxins are thought to be membrane receptors that bind proteins of the synaptobrevin/vesicle-associated membrane protein (VAMP) family found on transport vesicles. Recently, we detected synaptobrevin II and cellubrevin on immunopurified vesicles containing the glucose transporter 4 (GLUT4) in insulin-responsive cells. In an effort to identify the plasma membrane receptors for these vesicles, we now examine the expression of syntaxins in the 3T3-L1 adipocyte cell line. Neither syntaxin 1A nor 1B was found, in keeping with the neuronal restriction of these isoforms. In contrast, syntaxins 2 and 4 were readily detectable. By subcellular fractionation and estimation of protein yields, 67% of syntaxin 4 was localized to the plasma membrane, 24% to the low-density microsomes, and 9% to the high-density microsomes. Interestingly, acute insulin treatment decreased the content of syntaxin 4 in low-density microsomes and caused a corresponding gain in the plasma membrane fraction, reminiscent of the recruitment of GLUT4 glucose transporters. In contrast, there was no change in the distribution of syntaxin 2, which was mostly associated in the plasma membrane. A fraction of the intracellular syntaxin 4 was recovered with immunopurified GLUT4-containing vesicles. Moreover, anti-syntaxin 4 antibodies introduced in permeabilized 3T3-L1 adipocytes significantly reduced the insulin-dependent stimulation of glucose transport, in contrast to the introduction of irrelevant immunoglobulin G, which was without consequence. We propose that either the plasma membrane and/or the vesicular syntaxin 4 are involved in docking and/or fusion of GLUT4 vesicles at the cell surface of 3T3-L1 adipocytes.     

Syntaxin 6 regulates Glut4 trafficking in 3T3-L1 adipocytes

Insulin stimulates the movement of glucose transporter-4 (Glut4)-containing vesicles to the plasma membrane of adipose cells. We investigated the role of post-Golgi t-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in the trafficking of Glut4 in 3T3-L1 adipocytes. Greater than 85% of syntaxin 6 was found in Glut4-containing vesicles, and this t-SNARE exhibited insulin-stimulated movement to the plasma membrane. In contrast, the colocalization of Glut4 with syntaxin 7, 8, or 12/13 was limited and these molecules did not translocate to the plasma membrane. We used adenovirus to overexpress the cytosolic domain of these syntaxin's and studied their effects on Glut4 traffic. Overexpression of the cytosolic domain of syntaxin 6 did not affect insulin-stimulated glucose transport, but increased basal deGlc transport and cell surface Glut4 levels. Moreover, the syntaxin 6 cytosolic domain significantly reduced the rate of Glut4 reinternalization after insulin withdrawal and perturbed subendosomal Glut4 sorting; the corresponding domains of syntaxins 8 and 12 were without effect. Our data suggest that syntaxin 6 is involved in a membrane-trafficking step that sequesters Glut4 away from traffic destined for the plasma membrane. We speculate that this is at the level of traffic of Glut4 into its unique storage compartment and that syntaxin 16 may be involved.     

Role of SNAP23 in insulin-induced translocation of GLUT4 in 3T3-L1 adipocytes. Mediation of complex formation between syntaxin4 and VAMP2

Both syntaxin4 and VAMP2 are implicated in insulin regulation of glucose transporter-4 (GLUT4) trafficking in adipocytes as target (t) soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) and vesicle (v)-SNARE proteins, respectively, which mediate fusion of GLUT4-containing vesicles with the plasma membrane. Synaptosome-associated 23-kDa protein (SNAP23) is a widely expressed isoform of SNAP25, the principal t-SNARE of neuronal cells, and colocalizes with syntaxin4 in the plasma membrane of 3T3-L1 adipocytes. In the present study, two SNAP23 mutants, SNAP23-DeltaC8 (amino acids 1 to 202) and SNAP23-DeltaC49 (amino acids 1 to 161), were generated to determine whether SNAP23 is required for insulin-induced translocation of GLUT4 to the plasma membrane in 3T3-L1 adipocytes. Wild-type SNAP23 (SNAP23-WT) promoted the interaction between syntaxin4 and VAMP2 both in vitro and in vivo. Although SNAP23-DeltaC49 bound to neither syntaxin4 nor VAMP2, the SNAP23-DeltaC8 mutant bound to syntaxin4 but not to VAMP2. In addition, although SNAP23-DeltaC8 bound to syntaxin4, it did not mediate the interaction between syntaxin4 and VAMP2. Moreover, overexpression of SNAP23-DeltaC8 in 3T3-L1 adipocytes by adenovirus-mediated gene transfer inhibited insulin-induced translocation of GLUT4 but not that of GLUT1. In contrast, overexpression of neither SNAP23-WT nor SNAP23-DeltaC49 in 3T3-L1 adipocytes affected the translocation of GLUT4 or GLUT1. Together, these results demonstrate that SNAP23 contributes to insulin-dependent trafficking of GLUT4 to the plasma membrane in 3T3-L1 adipocytes by mediating the interaction between t-SNARE (syntaxin4) and v-SNARE (VAMP2).     

Protein kinase B stimulates the translocation of GLUT4 but not GLUT1 or transferrin receptors in 3T3-L1 adipocytes by a pathway involving SNAP-23, synaptobrevin-2, and/or cellubrevin.

An interaction of SNAP-23 and syntaxin 4 on the plasma membrane with vesicle-associated synaptobrevin-2 and/or cellubrevin, known as SNAP (soluble N-ethyl-maleimide-sensitive factor attachment protein) receptors or SNAREs, has been proposed to provide the targeting and/or fusion apparatus for insulin-stimulated translocation of the GLUT4 isoform of glucose transporter to the plasma membrane. By microinjecting 3T3-L1 adipocytes with the Clostridium botulinum toxin B or E, which proteolyzed synaptobrevin-2/cellubrevin and SNAP-23, respectively, we investigated the role of these SNAREs in GLUT4, GLUT1, and transferrin receptor trafficking. As expected, insulin stimulated the translocation of GLUT4, GLUT1, and transferrin receptors to the plasma membrane. By contrast, a constitutively active protein kinase B (PKB-DD) only stimulated a translocation of GLUT4 and not GLUT1 or the transferrin receptor. The GLUT4 response to PKB-DD was abolished by toxins B or E, whereas the insulin-evoked translocation of GLUT4 was inhibited by approximately 65%. These toxins had no significant effect on insulin-stimulated transferrin receptor appearance at the cell surface. Thus, insulin appears to induce GLUT4 translocation via two distinct routes, only one of which involves SNAP-23 and synaptobrevin-2/cellubrevin, and can be mobilized by PKB-DD. The PKB-, SNAP-23-, and synaptobrevin-2/cellubrevin-independent GLUT4 translocation pathway may involve movement through recycling endosomes, together with GLUT1 and transferrin receptors.     

Syntaxin 4, VAMP2, and/or VAMP3/cellubrevin are functional target membrane and vesicle SNAP receptors for insulin-stimulated GLUT4 translocation in adipocytes.

Introduction of the cytoplasmic domain of syntaxin 4, using either recombinant vaccinia virus or single-cell microinjection, resulted in an inhibition of insulin-stimulated GLUT4 but not GLUT1 translocation to the plasma membrane. This was specific for syntaxin 4, since neither the expression of syntaxin 3 nor the expression of a syntaxin 4 mutant in which the vesicle-associated membrane protein (VAMP) binding site was deleted had any significant effect. Consistent with the requirement for a functional VAMP binding site, expression of the cytoplasmic domains of VAMP2 or VAMP3/cellubrevin also resulted in an inhibition of insulin-stimulated GLUT4 translocation. In addition, immunoprecipitation of the expressed syntaxin 4 cytoplasmic domain resulted in an insulin-stimulated increase in the coimmunoprecipitation of GLUT4-containing vesicles. Together, these data demonstrate that syntaxin 4, VAMP2, and/or VAMP3/cellubrevin can function as target membrane and vesicle SNAP receptors, respectively, for insulin-responsive GLUT4 translocation to the plasma membrane.     

Glucosamine-induced insulin resistance is coupled to O-linked glycosylation of Munc18c

Evidence suggests that glucosamine inhibits distal components regulating insulin-stimulated GLUT4 translocation to the plasma membrane. Here we assessed whether key membrane docking and fusion events were targeted. Consistent with a plasma membrane-localized effect, 3T3-L1 adipocytes exposed to glucosamine displayed an increase in cell-surface O-linked glycosylation and a simultaneously impaired mobilization of GLUT4 by insulin. Analysis of syntaxin 4 and SNAP23, plasma membrane-localized target receptor proteins (t-SNAREs) for the GLUT4 vesicle, showed that they were not cell-surface targets of O-linked glycosylation. However, the syntaxin 4 binding protein, Munc18c, was targeted by O-linked glycosylation. This occurred concomitantly with a block in insulin-stimulated association of syntaxin 4 with its cognate GLUT4 vesicle receptor protein (v-SNARE), VAMP2. In conclusion, our data suggest that the mechanism by which glucosamine inhibits insulin-stimulated GLUT4 translocation involves modification of Munc18c.     

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.     

The t-SNAREs syntaxin4 and SNAP23 but not v-SNARE VAMP2 are indispensable to tether GLUT4 vesicles at the plasma membrane in adipocyte

SNARE proteins (VAMP2, syntaxin4, and SNAP23) have been thought to play a key role in GLUT4 trafficking by mediating the tethering, docking and subsequent fusion of GLUT4-containing vesicles with the plasma membrane. The precise functions of these proteins have remained elusive, however. We have now shown that depletion of the vesicle SNARE (v-SNARE) VAMP2 by RNA interference in 3T3-L1 adipocytes inhibited the fusion of GLUT4 vesicles with the plasma membrane but did not affect tethering of the vesicles to the membrane. In contrast, depletion of the target SNAREs (t-SNAREs) syntaxin4 or SNAP23 resulted in impairment of GLUT4 vesicle tethering to the plasma membrane. Our results indicate that the t-SNAREs syntaxin4 and SNAP23 are indispensable for the tethering of GLUT4 vesicles to the plasma membrane, whereas the v-SNARE VAMP2 is not required for this step but is essential for the subsequent fusion event.     

SNARE expression and distribution during 3T3-L1 adipocyte differentiation

Differentiation of 3T3-L1 cells into adipocytes presupposes the expression of the glucose transporter isoform GLUT4 and the acquisition of insulin-dependent GLUT4 translocation from intracellular storage vesicles to plasma membrane. This ability to translocate GLUT4 depends on the presence of a set of proteins of the SNARE category that are essential in the fusion step. The expression and levels of some of these SNARE proteins are altered during 3T3-L1 differentiation. Levels of the v-SNARE protein cellubrevin and of the t-SNARE protein syntaxin 4 were increased in this process in parallel to GLUT4. However, the levels of SNAP-23, another t-SNARE, were maintained during differentiation. Immunofluorescence images of SNAP-23 showed the initial distribution of this protein in a perinuclear region before differentiation and its redistribution towards plasma membrane in the adipocyte form. These results suggest a capital role in the expression levels and cellular distribution, during 3T3-L1 differentiation, of SNARE proteins involved in the late steps of GLUT4 translocation.     

Negative Regulation of Syntaxin4/SNAP-23/VAMP2-Mediated Membrane Fusion by Munc18c In Vitro

Background

Translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the plasma membrane is responsible for the increased rate of glucose transport into fat and muscle cells in response to insulin. This represents a specialised form of regulated membrane trafficking. Intracellular membrane traffic is subject to multiple levels of regulation by conserved families of proteins in all eukaryotic cells. Notably, all intracellular fusion events require SNARE proteins and Sec1p/Munc18 family members. Fusion of GLUT4-containing vesicles with the plasma membrane of insulin-sensitive cells involves the SM protein Munc18c, and is regulated by the formation of syntaxin 4/SNAP23/VAMP2 SNARE complexes.

Methodology/Principal Findings

Here we have used biochemical approaches to characterise the interaction(s) of Munc18c with its cognate SNARE proteins and to examine the role of Munc18c in regulating liposome fusion catalysed by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. We demonstrate that Munc18c makes contacts with both t- and v-SNARE proteins of this complex, and directly inhibits bilayer fusion mediated by the syntaxin 4/SNAP23/VAMP2 SNARE complex.

Conclusion/Significance

Our reductionist approach has enabled us to ascertain a direct inhibitory role for Munc18c in regulating membrane fusion mediated by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. It is important to note that two different SM proteins have recently been shown to stimulate liposome fusion mediated by their cognate SNARE complexes. Given the structural similarities between SM proteins, it seems unlikely that different members of this family perform opposing regulatory functions. Hence, our findings indicate that Munc18c requires a further level of regulation in order to stimulate SNARE-mediated membrane fusion.     

Thyroid hormone promotes insulin-induced glucose uptake by enhancing Akt phosphorylation and VAMP2 translocation in 3T3-L1 adipocytes

The purpose of this study was to test a hypothesis that T3 promotes glucose uptake via enhancing insulin-induced Akt phosphorylation and VAMP2 translocation in 3T3-L1 adipocytes. T3 significantly enhanced insulin-induced phosphorylation of Akt, cytoplasma to cell membrane translocations of vesicle-associated membrane protein 2 (VAMP2) and glucose transporter 4 (GLUT4), and glucose uptake in adipocytes. Akt inhibitor X abolished the promoting effects of T3, suggesting that Akt activation is essential for T3 to enhance these insulin-induced events in adipocytes. Knockdown of VAMP2 using siRNA abrogated the effects of T3 on insulin-induced GLUT4 translocation and glucose uptake, suggesting that VAMP2 is an important mediator of these processes. These data suggest that T3 may promote glucose uptake via enhancing insulin-induced phosphorylation of Akt and subsequent translocations of VAMP2 and GLUT4 in 3T3-L1 adipocytes. Akt phosphorylation is necessary for the promoting effects of T3 on insulin-stimulated VAMP2 translocation. Further, VAMP2 is essential for T3 to increase insulin-stimulated translocation of GLUT4 and subsequent uptake of glucose in adipocytes.     

Golgi-localized, gamma-ear-containing, Arf-binding protein adaptors mediate insulin-responsive trafficking of glucose transporter 4 in 3T3-L1 adipocytes

Small glucose transporter 4 (Glut4)-containing vesicles represent the major insulin-responsive compartment in fat and skeletal muscle cells. The molecular mechanism of their biogenesis is not yet elucidated. Here, we studied the role of the newly discovered family of monomeric adaptor proteins, GGA (Golgi-localized, gamma-ear-containing, Arf-binding proteins), in the formation of small Glut4 vesicles and acquisition of insulin responsiveness in 3T3-L1 adipocytes. In these cells, all three GGA isoforms are expressed throughout the differentiation process. In particular, GGA2 is primarily present in trans-Golgi network and endosomes where it demonstrates a significant colocalization with the recycling pool of Glut4. Using the techniques of immunoadsorption as well as glutathione-S-transferase pull-down assay we found that Glut4 vesicles (but not Glut4 per se) interact with GGA via the Vps-27, Hrs, and STAM (VHS) domain. Moreover, a dominant negative GGA mutant inhibits formation of Glut4 vesicles in vitro. To study a possible role of GGA in Glut4 traffic in the living cell, we stably expressed a dominant negative GGA mutant in 3T3-L1 adipocytes. Formation of small insulin-responsive Glut4-containing vesicles and insulin-stimulated glucose uptake in these cells were markedly impaired. Thus, GGA adaptors participate in the formation of the insulin-responsive vesicular compartment from the intracellular donor membranes both in vivo and in vitro.     

Syntaxin 16 controls the intracellular sequestration of GLUT4 in 3T3-L1 adipocytes

The regulated delivery of Glut4-containing vesicles to the plasma membrane is a specialised example of regulated membrane trafficking. Present models favour the transporter trafficking through two inter-related endosomal cycles. The first is the proto-typical endosomal system. This is a fast trafficking event that, in the absence of insulin, serves to internalise Glut4 from the plasma membrane. Once in this pathway, Glut4 is further sorted into a slowly recycling pathway that operates between recycling endosomes, the trans Golgi network, and a population of vesicles often referred to as Glut4-storage vesicles. Little is known about the molecules that regulate these distinct sorting steps. Here, we have studied the role of Stx16 in Glut4 trafficking. Using two independent strategies, we show that Stx16 plays a crucial role in Glut4 traffic in 3T3-L1 adipocytes. Over-expression of a mutant form of Stx16 devoid of a transmembrane anchor was found to significantly slow the reversal of insulin-stimulated glucose transport. Depletion of Stx16 using antisense approaches profoundly reduced insulin-stimulated glucose transport but was without effect on cell surface transferrin receptor levels, and also reduced the extent of Glut4 translocation to the plasma membrane in response to insulin. These data support a model in which Stx16 is crucial in the sorting of Glut4 from the fast cycling to the slow cycling intracellular trafficking pathways in adipocytes.     

Munc18-2/syntaxin3 complexes are spatially separated from syntaxin3-containing SNARE complexes

Exocytosis of mast cell granules requires a vesicular- and plasma membrane-associated fusion machinery. We examined the distribution of SNARE membrane fusion and Munc18 accessory proteins in lipid rafts of RBL mast cells. SNAREs were found either excluded (syntaxin2), equally distributed between raft and non-raft fractions (syntaxin4, VAMP-8, VAMP-2), or selectively enriched in rafts (syntaxin3, SNAP-23). Syntaxin4-binding Munc18-3 was absent, whereas small amounts of the syntaxin3-interacting partner Munc18-2 consistently distributed into rafts. Cognate SNARE complexes of syntaxin3 with SNAP-23 and VAMP-8 were enriched in rafts, whereas Munc18-2/syntaxin3 complexes were excluded. This demonstrates a spatial separation between these two types of complexes and suggests that Munc18-2 acts in a step different from SNARE complex formation and fusion.     

Resolving the function of distinct Munc18-1/SNARE protein interaction modes in a reconstituted membrane fusion assay

Sec1p/Munc18 proteins and SNAP receptors (SNAREs) are key components of the intracellular membrane fusion machinery. Compartment-specific v-SNAREs on a transport vesicle pair with their cognate t-SNAREs on the target membrane and drive lipid bilayer fusion. In a reconstituted assay that dissects the sequential assembly of t-SNARE (syntaxin 1·SNAP-25) and v-/t-SNARE (VAMP2·syntaxin 1·SNAP-25) complexes, and finally measures lipid bilayer merger, we resolved the inhibitory and stimulatory functions of the Sec1p/Munc18 protein Munc18-1 at the molecular level. Inhibition of membrane fusion by Munc18-1 requires a closed conformation of syntaxin 1. Remarkably, the concurrent preincubation of Munc18-1-inhibited syntaxin 1 liposomes with both VAMP2 liposomes and SNAP-25 at low temperature releases the inhibition and effectively stimulates membrane fusion. VAMP8 liposomes can neither release the inhibition nor exert the stimulatory effect, demonstrating the need for a specific Munc18-1/VAMP2 interaction. In addition, Munc18-1 binds to the N-terminal peptide of syntaxin 1, which is obligatory for a robust stimulation of membrane fusion. In contrast, this interaction is neither required for the inhibitory function of Munc18-1 nor for the release of this block. These results indicate that Munc18-1 and the neuronal SNAREs already have the inherent capability to function as a basic stage-specific off/on switch to control membrane fusion.     

The glucose transporter (GLUT-4) and vesicle-associated membrane protein-2 (VAMP-2) are segregated from recycling endosomes in insulin- sensitive cells

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.     

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.