Munc18c function is required for insulin-stimulated plasma membrane fusion of GLUT4 and insulin-responsive amino peptidase storage vesicles

To examine the functional role of the interaction between Munc18c and syntaxin 4 in the regulation of GLUT4 translocation in 3T3L1 adipocytes, we assessed the effects of introducing three different peptide fragments (20 to 24 amino acids) of Munc18c from evolutionarily conserved regions of the Sec1 protein family predicted to be solvent exposed. One peptide, termed 18c/pep3, inhibited the binding of full-length Munc18c to syntaxin 4, whereas expression of the other two peptides had no effect. In parallel, microinjection of 18c/pep3 but not a control peptide inhibited the insulin-stimulated translocation of endogenous GLUT4 and insulin-responsive amino peptidase (IRAP) to the plasma membrane. In addition, expression of 18c/pep3 prevented the insulin-stimulated fusion of endogenous and enhanced green fluorescent protein epitope-tagged GLUT4- and IRAP-containing vesicles into the plasma membrane, as assessed by intact cell immunofluorescence. However, unlike the pattern of inhibition seen with full-length Munc18c expression, cells expressing 18c/pep3 displayed discrete clusters of GLUT4 abd IRAP storage vesicles at the cell surface which were not contiguous with the plasma membrane. Together, these data suggest that the interaction between Munc18c and syntaxin 4 is required for the integration of GLUT4 and IRAP storage vesicles into the plasma membrane but is not necessary for the insulin-stimulated trafficking to and association with the cell surface.     

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.     

Munc18c interaction with syntaxin 4 monomers and SNARE complex intermediates in GLUT4 vesicle trafficking

In the process of insulin-stimulated GLUT4 vesicle exocytosis, Munc18c has been proposed to control SNARE complex formation by inactivating syntaxin 4 in a self-associated conformation. Using in vivo fluorescence resonance energy transfer in 3T3L1 adipocytes, co-immunoprecipitation, and in vitro binding assays, we provide data to indicate that Munc18c also associates with nearly equal affinity to a mutant of syntaxin 4 in a constitutively open (unfolded) state (L173A/E174A; LE). To bind to the open conformation of syntaxin 4, we found that Munc18c requires an interaction with the N terminus of syntaxin 4, which resembles Sly1 interaction with the N terminus of ER/Golgi syntaxins. However, both N and C termini of syntaxin 4 are required for Munc18c binding, since a mutation in the syntaxin 4 SNARE domain (I241A) reduces the interaction, irrespective of syntaxin 4 conformation. Using an optical reporter for syntaxin 4-SNARE pairings in vivo, we demonstrate that Munc18c blocks recruitment of SNAP23 to wild type syntaxin 4 yet associates with syntaxin 4LE-SNAP23 Q-SNARE complexes. Fluorescent imaging of GLUT4 vesicles in 3T3L1 adipocytes revealed that syntaxin 4LE expressed with Munc18c bypasses the requirement of insulin for GLUT4 vesicle plasma membrane docking. This effect was attenuated by reducing the Munc18c-syntaxin 4LE interaction with the I241A mutation, indicating that Munc18c facilitates vesicle docking. Therefore, in contradiction to previous models, our data indicates that the conformational "opening" of syntaxin 4 rather than the dissociation of Munc18c is the critical event required for GLUT4 vesicle docking.     

Munc18c regulates insulin-stimulated glut4 translocation to the transverse tubules in skeletal muscle

To examine the intracellular trafficking and translocation of GLUT4 in skeletal muscle, we have generated transgenic mouse lines that specifically express a GLUT4-EGFP (enhanced green fluorescent protein) fusion protein under the control of the human skeletal muscle actin promoter. These transgenic mice displayed EGFP fluorescence restricted to skeletal muscle and increased glucose tolerance characteristic of enhanced insulin sensitivity. The GLUT4-EGFP protein localized to the same intracellular compartment as the endogenous GLUT4 protein and underwent insulin- and exercise-stimulated translocation to both the sarcolemma and transverse-tubule membranes. Consistent with previous studies in adipocytes, overexpression of the syntaxin 4-binding Munc18c isoform, but not the related Munc18b isoform, in vivo specifically inhibited insulin-stimulated GLUT4-EGFP translocation. Surprisingly, however, Munc18c inhibited GLUT4 translocation to the transverse-tubule membrane without affecting translocation to the sarcolemma membrane. The ability of Munc18c to block GLUT4-EGFP translocation to the transverse-tubule membrane but not the sarcolemma membrane was consistent with substantially reduced levels of syntaxin 4 in the transverse-tubule membrane. Together, these data demonstrate that Munc18c specifically functions in the compartmentalized translocation of GLUT4 to the transverse-tubules in skeletal muscle. In addition, these results underscore the utility of this transgenic model to directly visualize GLUT4 translocation in skeletal muscle.     

Temporal separation of insulin-stimulated GLUT4/IRAP vesicle plasma membrane docking and fusion in 3T3L1 adipocytes

Examination of the time and temperature dependence of insulin-stimulated GLUT4/IRAP-containing vesicle trafficking demonstrated an approximate 7-fold increase in the half-time for plasma membrane translocation at 23 degrees C (t((1)/(2)) = approximately 30 min) compared with 37 degrees C (t((1)/(2)) = approximately 4 min) without a significant change in the extent of either GLUT4 or IRAP translocation. Localization of the endogenous GLUT4 and expressed GLUT4-enhanced green fluorescent protein fusion protein in intact 3T3L1 adipocytes demonstrated that at 23 degrees C there was a time-dependent accumulation of discrete GLUT4-containing vesicles adjacent to the inner face of the cell surface membrane but that was not contiguous and/or physically incorporated into the plasma membrane. Together, these data demonstrate that the temperature-dependent decrease in the rate of GLUT4 and IRAP translocation results from a reduction in GLUT4/IRAP-containing vesicle fusion and not trafficking or docking to the plasma membrane.     

Cellular munc18c levels can modulate glucose transport rate and GLUT4 translocation in 3T3L1 cells

Munc18c has been shown to bind syntaxin 4 and to play a role in GLUT4 translocation and glucose transport, although this role is as yet poorly defined. In the present study, the effects of modulating the available level of munc18c on glucose transport and GLUT4 translocation were examined. Over-expression of munc18c in 3T3L1 adipocytes inhibited insulin-stimulated glucose transport by approximately 50%. Basal glucose transport rates were also decreased by approximately 25%. In contrast, microinjection of a munc18c polyclonal antibody stimulated GLUT4 translocation by approximately 60% over basal levels without affecting insulin-stimulated GLUT4 levels. Microinjection of a control antibody had no effect. These data are consistent with the likelihood that antibody microinjection sequesters munc18c enabling translocation/fusion of GLUT4 vesicles. Mutagenesis of a potential proline-directed kinase phosphorylation site in munc18c, T569, that in previous studies of its neuronal counterpart munc18a caused its dissociation from its complex with syntaxin 1a, had no effect on munc18c's association with syntaxin 4 or its inhibition of glucose transport, indicative that phosphorylation of this residue is not important for insulin regulation of glucose transport. The over-expression and microinjection sequestration data support an inhibitory role for munc18c on translocation/fusion of GLUT4 vesicles. They further show that altering the level of available munc18c in 3T3L1 cells can modulate glucose transport rates, indicating its potential as a target for therapeutics in diabetes.     

Munc18c phosphorylation by the insulin receptor links cell signaling directly to SNARE exocytosis

How the Sec1/Munc18-syntaxin complex might transition to form the SNARE core complex remains unclear. Toward this, Munc18c tyrosine phosphorylation has been correlated with its dissociation from syntaxin 4. Using 3T3-L1 adipocytes subjected to small interfering ribonucleic acid reduction of Munc18c as a model of impaired insulin-stimulated GLUT4 vesicle exocytosis, we found that coordinate expression of Munc18c-wild type or select phosphomimetic Munc18c mutants, but not phosphodefective mutants, restored GLUT4 vesicle exocytosis, suggesting a requirement for Munc18c tyrosine phosphorylation at Tyr219 and Tyr521. Surprisingly, the insulin receptor (IR) tyrosine kinase was found to target Munc18c at Tyr521 in vitro, rapidly binding and phosphorylating endogenous Munc18c within adipocytes and skeletal muscle. IR, but not phosphatidylinositol 3-kinase, activation was required. Altogether, we identify IR as the first known tyrosine kinase for Munc18c as part of a new insulin-signaling step in GLUT4 vesicle exocytosis, exemplifying a new model for the coordination of SNARE assembly and vesicle mobilization events in response to a single extracellular stimulus.     

Munc18c provides stimulus-selective regulation of GLUT4 but not fatty acid transporter trafficking in skeletal muscle

Insulin-, and contraction-induced GLUT4 and fatty acid (FA) transporter translocation may share common trafficking mechanisms. Our objective was to examine the effects of partial Munc18c ablation on muscle glucose and FA transport, FA oxidation, GLUT4 and FA transporter (FAT/CD36, FABPpm, FATP1, FATP4) trafficking to the sarcolemma, and FAT/CD36 to mitochondria. In Munc18c(-/+) mice, insulin-stimulated glucose transport and GLUT4 sarcolemmal appearance were impaired, but were unaffected by contraction. Insulin- and contraction-stimulated FA transport, sarcolemmal FA transporter appearance, and contraction-mediated mitochondrial FAT/CD36 were increased normally in Munc18c(-/+) mice. Hence, Munc18c provides stimulus-specific regulation of GLUT4 trafficking, but not FA transporter trafficking.     

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.     

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.     

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.     

Tomosyn interacts with the t-SNAREs syntaxin4 and SNAP23 and plays a role in insulin-stimulated GLUT4 translocation

The Sec1p-like/Munc18 (SM) protein Munc18a binds to the neuronal t-SNARE Syntaxin1A and inhibits SNARE complex assembly. Tomosyn, a cytosolic Syntaxin1A-binding protein, is thought to regulate the interaction between Syntaxin1A and Munc18a, thus acting as a positive regulator of SNARE assembly. In the present study we have investigated the interaction between b-Tomosyn and the adipocyte SNARE complex involving Syntaxin4/SNAP23/VAMP-2 and the SM protein Munc18c, in vitro, and the potential involvement of Tomosyn in regulating the translocation of GLUT4 containing vesicles, in vivo. Tomosyn formed a high affinity ternary complex with Syntaxin4 and SNAP23 that was competitively inhibited by VAMP-2. Using a yeast two-hybrid assay we demonstrate that the VAMP-2-like domain in Tomosyn facilitates the interaction with Syntaxin4. Overexpression of Tomosyn in 3T3-L1 adipocytes inhibited the translocation of green fluorescent protein-GLUT4 to the plasma membrane. The SM protein Munc18c was shown to interact with the Syntaxin4 monomer, Syntaxin4 containing SNARE complexes, and the Syntaxin4/Tomosyn complex. These data suggest that Tomosyn and Munc18c operate at a similar stage of the Syntaxin4 SNARE assembly cycle, which likely primes Syntaxin4 for entry into the ternary SNARE complex.     

脂肪细胞中Munc18c的缺失不影响胰岛素诱导的GLUT4转运(已撤稿2016-01-13)

动物脂肪和肌肉组织中葡萄糖的摄取是通过受胰岛素调控的GLUT4储存囊泡的运输实现的.Sec1p的同源物Munc18c被认为是通过控制SNARE复合物的装配来使GLUT4囊泡锚定到质膜上的重要物质.我们发现Munc18c的缺失没有影响GLUT4的转运上膜,也没有影响Syntaxin4在细胞膜上的定位.在缺少Munc18c和功能性Syntaxin2的时候,GLUT4的转运可能和Munc18b有关.在3T3-L1脂肪细胞中与Syntaxin4具有强烈相互作用的是Munc18c而不是Munc18a和Munc18b.然而,当缺少Munc18c时,Munc18a和Munc18b与Syntaxin4体现出较弱的相互作用.因此,Syntaxin4可能在胰岛素刺激GLUT4转运过程中起到重要的作用,且与SM蛋白的相互作用是有代偿性的.     

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.     

A specific dileucine motif is required for the GGA-dependent entry of newly synthesized insulin-responsive aminopeptidase into the insulin-responsive compartment

In muscle and adipose cells, the insulin-responsive aminopeptidase (IRAP) is localized to intracellular storage sites and undergoes insulin-dependent redistribution to the cell surface. Following expression, the newly synthesized IRAP protein traffics to the perinuclear insulin-sensitive compartment and acquires insulin sensitivity 6-9 h following biosynthesis. Knockdown of GGA1 by RNA interference prevented IRAP from entering, but not exiting, the insulin-responsive compartment. Mutation of the dileucine motif at positions 76 and 77 (EGFP-IRAP/AA(76,77)), but not the dileucine motif at positions 53 and 54, resulted in the rapid default of the reporter to the cell surface beginning at 3 h following biosynthesis. Alanine substitution of 9 residues amino- or carboxyl-terminal to LL(76,77) did not perturb basal intracellular sequestration or abrogate insulin-stimulated IRAP translocation. Moreover, a dominant interfering GGA mutant (VHS-GAT) potently inhibited insulin-stimulated translocation of EGFP-IRAP/WT but did not block the constitutive exocytotic trafficking of EGFP-IRAP/AA(76,77). In addition, the EGFP-IRAP/WT and EGFP-IRAP/AA(76,77) constructs occupied morphologically distinct tubulovesicular compartments in the perinuclear region. Taken together, these data indicate that LL(76,77) functions during the GGA-dependent sorting of newly made IRAP into the insulin-responsive storage compartment.     

GLUT4 ablation in mice results in redistribution of IRAP to the plasma membrane

Glucose transporter (GLUT) 4 is the insulin responsive glucose transporter in adipose tissue, skeletal muscle, and heart. Insulin elicits increased glucose uptake by recruiting GLUT4 from a specialized intracellular storage site to the cell surface. Expression of various proteins that colocalize with GLUT4 and/or are involved in insulin-stimulated GLUT4 translocation was examined in adipocytes as well as skeletal and cardiac muscles from GLUT4 null mice. Our data demonstrate that expression of insulin-regulated aminopeptidase (IRAP) is divergently regulated in GLUT4 null tissues, e.g., upregulated 1.6-fold in GLUT4 null adipocytes and downregulated in GLUT4 null skeletal muscle (40%) and heart (60%). IRAP exhibited abnormal subcellular distribution and impaired insulin-stimulated translocation in GLUT4-deficient tissues. We propose the compartment containing IRAP and proteins normally associated with GLUT4 vesicle traffics constitutively to the cell surface in GLUT4 null adipocytes and skeletal muscle.     

Molecular dissection of the Munc18c/syntaxin4 interaction: implications for regulation of membrane trafficking

Sec1p/Munc18 (SM) proteins are believed to play an integral role in vesicle transport through their interaction with SNAREs. Different SM proteins have been shown to interact with SNAREs via different mechanisms, leading to the conclusion that their function has diverged. To further explore this notion, in this study, we have examined the molecular interactions between Munc18c and its cognate SNAREs as these molecules are ubiquitously expressed in mammals and likely regulate a universal plasma membrane trafficking step. Thus, Munc18c binds to monomeric syntaxin4 and the N-terminal 29 amino acids of syntaxin4 are necessary for this interaction. We identified key residues in Munc18c and syntaxin4 that determine the N-terminal interaction and that are consistent with the N-terminal binding mode of yeast proteins Sly1p and Sed5p. In addition, Munc18c binds to the syntaxin4/SNAP23/VAMP2 SNARE complex. Pre-assembly of the syntaxin4/Munc18c dimer accelerates the formation of SNARE complex compared to assembly with syntaxin4 alone. These data suggest that Munc18c interacts with its cognate SNAREs in a manner that resembles the yeast proteins Sly1p and Sed5p rather than the mammalian neuronal proteins Munc18a and syntaxin1a. The Munc18c-SNARE interactions described here imply that Munc18c could play a positive regulatory role in SNARE assembly.     

Insulin-stimulated exocytosis of GLUT4 is enhanced by IRAP and its partner tankyrase

The glucose transporter GLUT4 and the aminopeptidase IRAP (insulin-responsive aminopeptidase) are the major cargo proteins of GSVs (GLUT4 storage vesicles) in adipocytes and myocytes. In the basal state, most GSVs are sequestered in perinuclear and other cytosolic compartments. Following insulin stimulation, GSVs undergo exocytic translocation to insert GLUT4 and IRAP into the plasma membrane. The mechanisms regulating GSV trafficking are not fully defined. In the present study, using 3T3-L1 adipocytes transfected with siRNAs (small interfering RNAs), we show that insulin-stimulated IRAP translocation remained intact despite substantial GLUT4 knockdown. By contrast, insulin-stimulated GLUT4 translocation was impaired upon IRAP knockdown, indicating that IRAP plays a role in GSV trafficking. We also show that knockdown of tankyrase, a Golgi-associated IRAP-binding protein that co-localizes with perinuclear GSVs, attenuated insulin-stimulated GSV translocation and glucose uptake without disrupting insulin-induced phosphorylation cascades. Moreover, iodixanol density gradient analyses revealed that tankyrase knockdown altered the basal-state partitioning of GLUT4 and IRAP within endosomal compartments, apparently by shifting both proteins toward less buoyant compartments. Importantly, the afore-mentioned effects of tankyrase knockdown were reproduced by treating adipocytes with PJ34, a general PARP (poly-ADP-ribose polymerase) inhibitor that abrogated tankyrase-mediated protein modification known as poly-ADP-ribosylation. Collectively, these findings suggest that physiological GSV trafficking depends in part on the presence of IRAP in these vesicles, and that this process is regulated by tankyrase and probably its PARP activity.     

Syntaxin 4 expression affects glucose transporter 8 translocation and embryo survival

Target-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) are receptors that facilitate vesicle and target membrane fusion. Syntaxin 4 is the t-SNARE critical for insulin-stimulated glucose transporter 4 (GLUT4)-plasma membrane fusion in adipocytes. GLUT8 is a novel IGF-I/insulin-regulated glucose transporter expressed in the mouse blastocyst. Similar to GLUT4, GLUT8 translocates to the plasma membrane to increase glucose uptake at a stage in development when glucose serves as the main substrate. Any decrease in GLUT8 cell surface expression results in increased apoptosis and pregnancy loss. Previous studies have also shown that disruption of the syntaxin 4 (Stx4a) gene results in early embryonic lethality before embryonic d 7.5. We have now demonstrated that syntaxin 4 protein is localized predominantly to the apical plasma membrane of the murine blastocyst. Stx4a inheritance, as detected by protein expression, occurs with the expected Mendelian frequency up to embryonic d 4.5. In parallel, 22% of the blastocysts from Stx4a+/- matings had no significant insulin-stimulated translocation of GLUT8 whereas 77% displayed either partial or complete translocation to the apical plasma membrane. This difference in GLUT8 translocation directly correlated with one-third of blastocysts from Stx4a+/- mating having reduced rates of insulin-stimulated glucose uptake and 67% with wild-type rates. These data demonstrate that the lack of syntaxin 4 expression results in abnormal movement of GLUT8 in response to insulin, decreased insulin-stimulated glucose uptake, and increased apoptosis. Thus, syntaxin 4 functions as the necessary t-SNARE protein responsible for correct fusion of the GLUT8-containing vesicle with the plasma membrane in the mouse blastocyst.