Phosphorylation of Syntaxin 4 by the Insulin Receptor Drives Exocytic SNARE Complex Formation to Deliver GLUT4 to the Cell Surface.

A major consequence of insulin binding its receptor on fat and muscle cells is the stimulation of glucose transport into these tissues. This is achieved through an increase in the exocytic trafficking rate of the facilitative glucose transporter GLUT4 from intracellular stores to the cell surface. Delivery of GLUT4 to the cell surface requires the formation of functional SNARE complexes containing Syntaxin 4, SNAP23, and VAMP2. Insulin stimulates the formation of these complexes and concomitantly causes phosphorylation of Syntaxin 4. Here, we use a combination of biochemistry and cell biological approaches to provide a mechanistic link between these observations. We present data to support the hypothesis that Tyr-115 and Tyr-251 of Syntaxin 4 are direct substrates of activated insulin receptors, and that these residues modulate the protein's conformation and thus regulate the rate at which Syntaxin 4 forms SNARE complexes that deliver GLUT4 to the cell surface. This report provides molecular details on how the cell regulates SNARE-mediated membrane traffic in response to an external stimulus.

Large scale, single-cell FRET-based glucose uptake measurements within heterogeneous populations.

Fluorescent biosensors are powerful tools allowing the concentration of metabolites and small molecules, and other properties such as pH and molecular crowding to be measured inside live single cells. The technology has been hampered by lack of simple software to identify cells and quantify biosensor signals in single cells. We have developed a new software package, FRETzel, to address this gap and demonstrate its use by measuring insulin-stimulated glucose uptake in individual fat cells of varying sizes for the first time. Our results support the long-standing hypothesis that larger fat cells are less sensitive to insulin than smaller ones, a finding that has important implications for the battle against type 2 diabetes. FRETzel has been optimized using the messy and crowded environment of cultured adipocytes, demonstrating its utility for quantification of FRET biosensors in a wide range of other cell types, including fibroblasts and yeast via a simple user-friendly quantitative interface.

Knockout of syntaxin-4 in 3T3-L1 adipocytes reveals new insight into GLUT4 trafficking and adiponectin secretion.

Adipocytes are key to metabolic regulation, exhibiting insulin-stimulated glucose transport that is underpinned by the insulin-stimulated delivery of glucose transporter type 4 (SLC2A4, also known and hereafter referred to as GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, and increase cell surface GLUT4 levels. Adipocytokines, such as adiponectin, are secreted via a similar mechanism. We used genome editing to knock out syntaxin-4, a protein reported to mediate fusion between GLUT4-containing vesicles and the plasma membrane in 3T3-L1 adipocytes. Syntaxin-4 knockout reduced insulin-stimulated glucose transport and adiponectin secretion by ∼50% and reduced GLUT4 levels. Ectopic expression of haemagglutinin (HA)-tagged GLUT4 conjugated to GFP showed that syntaxin-4-knockout cells retain significant GLUT4 translocation capacity, demonstrating that syntaxin-4 is dispensable for insulin-stimulated GLUT4 translocation. Analysis of recycling kinetics revealed only a modest reduction in the exocytic rate of GLUT4 in knockout cells, and little effect on endocytosis. These analyses demonstrate that syntaxin-4 is not always rate limiting for GLUT4 delivery to the cell surface. In sum, we show that syntaxin-4 knockout results in reduced insulin-stimulated glucose transport, depletion of cellular GLUT4 levels and inhibition of adiponectin secretion but has only modest effects on the translocation capacity of the cells. This article has an associated First Person interview with Hannah L. Black and Rachel Livingstone, joint first authors of the paper.

CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis.

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.

The deubiquitinating enzyme USP25 binds tankyrase and regulates trafficking of the facilitative glucose transporter GLUT4 in adipocytes.

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.

Proximity Ligation Assay to Study the GLUT4 Membrane Trafficking Machinery.

In this chapter a detailed protocol of proximity ligation assay (PLA) is described thoroughly. PLA is a technique that allows detection of protein associations in situ, providing a sensitive and selective approach for protein-protein interaction studies. We demonstrate the technique by applying it for trafficking studies of the facilitative glucose transporter GLUT4. Trafficking of GLUT4 from perinuclear depots to the plasma membrane is regulated by insulin in adipocytes and muscle cells, and mediated by formation of functional SNARE complexes containing Syntaxin4, SNAP23, and VAMP2. The Sec1/Munc18 (SM) protein Munc18c also plays a key role in insulin-stimulated GLUT4 translocation via a series of different interactions with the SNARE complex and/or with the SNARE proteins individually. Studying the interactions that occur between SNARE proteins themselves and also with Munc18c in insulin-responsive cells is critical to further understand SNARE protein function and GLUT4 trafficking mechanism in general.

Alternative routes to the cell surface underpin insulin-regulated membrane trafficking of GLUT4.

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.

Studies of the regulated assembly of SNARE complexes in adipocytes.

Insulin plays a fundamental role in whole-body glucose homeostasis. Central to this is the hormone's ability to rapidly stimulate the rate of glucose transport into adipocytes and muscle cells [1]. Upon binding its receptor, insulin stimulates an intracellular signalling cascade that culminates in redistribution of glucose transporter proteins, specifically the GLUT4 isoform, from intracellular stores to the plasma membrane, a process termed 'translocation' [1,2]. This is an example of regulated membrane trafficking [3], a process that also underpins other aspects of physiology in a number of specialized cell types, for example neurotransmission in brain/neurons and release of hormone-containing vesicles from specialized secretory cells such as those found in pancreatic islets. These processes invoke a number of intriguing biological questions as follows. How is the machinery involved in these membrane trafficking events mobilized in response to a stimulus? How do the signalling pathways that detect the external stimulus interface with the trafficking machinery? Recent studies of insulin-stimulated GLUT4 translocation offer insight into such questions. In the present paper, we have reviewed these studies and draw parallels with other regulated trafficking systems.

Insulin stimulates syntaxin4 SNARE complex assembly via a novel regulatory mechanism.

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.

Arabidopsis Sec1/Munc18 protein SEC11 is a competitive and dynamic modulator of SNARE binding and SYP121-dependent vesicle traffic.

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.

Endosomal sorting of GLUT4 and Gap1 is conserved between yeast and insulin-sensitive cells.

The insulin-regulated trafficking of the facilitative glucose transporter GLUT4 in human fat and muscle cells and the nitrogen-regulated trafficking of the general amino acid permease Gap1 in the yeast Saccharomyces cerevisiae share several common features: Both Gap1 and GLUT4 are nutrient transporters that are mobilised to the cell surface from an intracellular store in response to an environmental cue; both are polytopic membrane proteins harbouring amino acid targeting motifs in their C-terminal tails that are required for their regulated trafficking; ubiquitylation of both Gap1 and GLUT4 plays an important role in their regulated trafficking, as do the ubiquitin-binding GGA (Golgi-localised, γ-ear-containing, ARF-binding) adaptor proteins. Here, we find that when expressed heterologously in yeast, human GLUT4 is subject to nitrogen-regulated trafficking in an ubiquitin-dependent manner similar to Gap1. In addition, by expressing a GLUT4/Gap1 chimeric protein in adipocytes we show that the carboxy-tail of Gap1 directs intracellular sequestration and insulin-regulated trafficking in adipocytes. These findings demonstrate that the trafficking signals and their cognate molecular regulatory machinery that mediate regulated exocytosis of membrane proteins are conserved across evolution.

Functional homology of mammalian syntaxin 16 and yeast Tlg2p reveals a conserved regulatory mechanism.

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 tlg2Delta 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.