The Arabidopsis thaliana Qa-SNARE SYP121 (=SYR1/PEN1) drives vesicle traffic at the plasma membrane of cells throughout the vegetative plant. It facilitates responses to drought, to the water stress hormone abscisic acid, and to pathogen attack, and it is essential for recovery from so-called programmed stomatal closure. How SYP121-mediated traffic is regulated is largely unknown, although it is thought to depend on formation of a fusion-competent SNARE core complex with the cognate partners VAMP721 and SNAP33. Like SYP121, the Arabidopsis Sec1/Munc18 protein SEC11 (=KEULE) is expressed throughout the vegetative plant. We find that SEC11 binds directly with SYP121 both in vitro and in vivo to affect secretory traffic. Binding occurs through two distinct modes, one requiring only SEC11 and SYP121 and the second dependent on assembly of a complex with VAMP721 and SNAP33. SEC11 competes dynamically for SYP121 binding with SNAP33 and VAMP721, and this competition is predicated by SEC11 association with the N terminus of SYP121. These and additional data are consistent with a model in which SYP121-mediated vesicle fusion is regulated by an unusual "handshaking" mechanism of concerted SEC11 debinding and rebinding. They also implicate one or more factors that alter or disrupt SEC11 association with the SYP121 N terminus as an early step initiating SNARE complex formation.

Glucose transporter 4 (GLUT4) is sequestered inside muscle and fat and then released by vesicle traffic to the cell surface in response to postprandial insulin for blood glucose clearance. Here, we map the biogenesis of this GLUT4 traffic pathway in humans, which involves clathrin isoform CHC22. We observe that GLUT4 transits through the early secretory pathway more slowly than the constitutively secreted GLUT1 transporter and localize CHC22 to the ER-to-Golgi intermediate compartment (ERGIC). CHC22 functions in transport from the ERGIC, as demonstrated by an essential role in forming the replication vacuole of Legionella pneumophila bacteria, which requires ERGIC-derived membrane. CHC22 complexes with ERGIC tether p115, GLUT4, and sortilin, and downregulation of either p115 or CHC22, but not GM130 or sortilin, abrogates insulin-responsive GLUT4 release. This indicates that CHC22 traffic initiates human GLUT4 sequestration from the ERGIC and defines a role for CHC22 in addition to retrograde sorting of GLUT4 after endocytic recapture, enhancing pathways for GLUT4 sequestration in humans relative to mice, which lack CHC22.

Insulin stimulates glucose transport into fat and muscle cells by increasing the exocytic trafficking rate of the GLUT4 facilitative glucose transporter from intracellular stores to the plasma membrane. Delivery of GLUT4 to the plasma membrane is mediated by formation of functional SNARE complexes containing syntaxin4, SNAP23, and VAMP2. Here we have used an in situ proximity ligation assay to integrate these two observations by demonstrating for the first time that insulin stimulation causes an increase in syntaxin4-containing SNARE complex formation in adipocytes. Furthermore, we demonstrate that insulin brings about this increase in SNARE complex formation by mobilizing a pool of syntaxin4 held in an inactive state under basal conditions. Finally, we have identified phosphorylation of the regulatory protein Munc18c, a direct target of the insulin receptor, as a molecular switch to coordinate this process. Hence, this report provides molecular detail of how the cell alters membrane traffic in response to an external stimulus, in this case, insulin.A major consequence of insulin binding its receptor on fat and muscle cells is a change in localization of the GLUT4 facilitative glucose transporter. In the absence of insulin, ϳ95% of the transporter is retained intracellularly. Activation of the insulin receptor tyrosine kinase culminates in a 10-to 20-fold increase in the amount of GLUT4 present at the cell surface, accounting for the increased rate of glucose transport into these cells (1). GLUT4 is sequestered away from the cell surface in the absence of insulin by continually cycling through two interrelated endosomal cycles (1). The first operates between the plasma membrane and early and recycling endosomes. This is a fast-trafficking loop that keeps steady-state levels of transporter at the cell surface low under basal conditions. GLUT4 is further sorted from this cycle into a more slowly operating loop between recycling endosomes, the transGolgi network, and a population of vesicles termed GSVs (GLUT4 storage vesicles). It is from GSVs that GLUT4 is mobilized to the cell surface in response to insulin (1).Like all eukaryotic membrane trafficking events, insulin-regulated trafficking of GLUT4 is mediated by formation of specific SNARE complexes. Cell surface delivery of GLUT4 is mediated by a SNARE complex containing the plasma membrane-localized syntaxin4 (Sx4) and SNAP23 t-SNAREs and the VAMP2 v-SNARE (2). SNARE complex formation contributes to specificity of membrane traffic and also provides energy for bilayer fusion (3). Thus, regulating SNARE complex assembly allows the cell to regulate membrane traffic, but whether insulin stimulates SNARE complex formation remains unknown. Sec1/Munc18 (SM) proteins are key regulators of membrane traffic exerting their effect through their cognate SNARE proteins, but their precise mode of action is not understood (3). The Munc18c SM protein binds to Sx4 and is required for insulin-regulated delivery of GLUT4 to the surface of fat and muscle cells (2). Intriguingly, M...

Glucose Transporter 4 (GLUT4) is sequestered inside muscle and fat, then released by vesicle traffic to the cell surface in response to post-prandial insulin for blood glucose clearance. Here we map the biogenesis of this GLUT4 traffic pathway in humans, which involves clathrin isoform CHC22. We observe that GLUT4 transits through the early secretory pathway more slowly than the constitutively-secreted GLUT1 transporter and localize CHC22 to the endoplasmic-reticulum-to-Golgi-intermediate compartment (ERGIC). CHC22 functions in transport from the ERGIC, as demonstrated by an essential role in forming the replication vacuole of Legionella pneumophila bacteria, which requires ERGIC-derived membrane. CHC22 complexes with ERGIC tether p115, GLUT4 and sortilin and down-regulation of either p115 or CHC22, but not GM130 or sortilin abrogate insulin-responsive GLUT4 release. This indicates CHC22 traffic initiates human GLUT4 sequestration from the ERGIC, and defines a role for CHC22 in addition to retrograde sorting of GLUT4 after endocytic recapture, enhancing pathways for GLUT4 sequestration in humans relative to mice, which lack CHC22.SummaryBlood glucose clearance relies on insulin-mediated exocytosis of glucose transporter 4 (GLUT4) from sites of intracellular sequestration. We show that in humans, CHC22 clathrin mediates membrane traffic from the ER-to-Golgi Intermediate Compartment, which is needed for GLUT4 sequestration during GLUT4 pathway biogenesis.

Key to whole body glucose homeostasis is the ability of fat and muscle cells to sequester the facilitative glucose transporter GLUT4 in an intracellular compartment from where it can be mobilized in response to insulin. We have previously demonstrated that this process requires ubiquitination of GLUT4 while numerous other studies have identified several molecules that are also required, including the insulin-responsive aminopeptidase IRAP and its binding partner, the scaffolding protein tankyrase. In addition to binding IRAP, Tankyrase has also been shown to bind the deubiquinating enzyme USP25. Here we demonstrate that USP25 and Tankyrase interact, and colocalise with GLUT4 in insulin-sensitive cells. Furthermore depletion of USP25 from adipocytes reduces cellular levels of GLUT4 and concomitantly blunts the ability of insulin to stimulate glucose transport. Collectively, these data support our model that sorting of GLUT4 into its insulin-sensitive store involves a cycle of ubiquitination and subsequent deubiquitination.

Insulin-stimulated delivery of glucose transporters (GLUT4, also known as SLC2A4) from specialized intracellular GLUT4 storage vesicles (GSVs) to the surface of fat and muscle cells is central to whole-body glucose regulation. This translocation and subsequent internalization of GLUT4 back into intracellular stores transits through numerous small membrane-bound compartments (internal GLUT4-containing vesicles; IGVs) including GSVs, but the function of these different compartments is not clear. Cellugyrin (also known as synaptogyrin-2) and sortilin define distinct populations of IGV; sortilin-positive IGVs represent GSVs, but the function of cellugyrin-containing IGVs is unknown. Here, we demonstrate a role for cellugyrin in intracellular sequestration of GLUT4 in HeLa cells and have used a proximity ligation assay to follow changes in pairwise associations between cellugyrin, sortilin, GLUT4 and membrane trafficking machinery following insulin-stimulation of 3T3-L1 adipoctyes. Our data suggest that insulin stimulates traffic from cellugyrin-containing to sortilin-containing membranes, and that cellugyrin-containing IGVs provide an insulin-sensitive reservoir to replenish GSVs following insulin-stimulated exocytosis of GLUT4. Furthermore, our data support the existence of a pathway from cellugyrin-containing membranes to the surface of 3T3-L1 adipocytes that bypasses GSVs under basal conditions, and that insulin diverts traffic away from this into GSVs.

Membrane fusion in all eukaryotic cells is regulated by the formation of specific SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes. The molecular mechanisms that control this process are conserved through evolution and require several protein families, including Sec1p/Munc18 (SM) proteins. Here, we demonstrate that the mammalian SNARE protein syntaxin 16 (Sx16, also known as Syn16) is a functional homologue of the yeast SNARE Tlg2p, in that its expression fully complements the mutant phenotypes of tlg2Δ mutant yeast. We have used this functional homology to demonstrate that, as observed for Tlg2p, the function of Sx16 is regulated by the SM protein Vps45p. Furthermore, in vitro SNARE-complex assembly studies demonstrate that the N-terminal domain of Tlg2p is inhibitory to the formation of SNARE complexes, and that this inhibition can be lifted by the addition of purified Vps45p. By combining these cell-biological and biochemical analyses, we propose an evolutionarily conserved regulatory mechanism for Vps45p function. Our data support a model in which the SM protein is required to facilitate a switch of Tlg2p and Sx16 from a closed to an open conformation, thus allowing SNARE-complex assembly and membrane fusion to proceed.

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