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

Insulin-stimulated delivery of glucose transporters (GLUT4) from specialized intracellular GLUT4 storage vesicles (GSVs) to the surface of fat and muscle cells is central to whole-body glucose. This translocation and subsequent internalization of GLUT4 back into intracellular stores transits numerous small membrane-bound compartments (internal GLUT4-containing vesicles; IGVs) including GSVs, but the function of these different compartments is not clear. Cellugyrin 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- to sortilin- membranes, and that cellugyrin-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-membranes to the surface of 3T3-L1 adipocytes that bypasses GSVs under basal conditions, and that insulin diverts traffic away from this into GSVs

The Sec1p/Munc18 (SM) protein, Vps45p, cycles on and off membranes during vesicle transport

Protein phosphatase 1 (PP1, Glc7p) functions in the final stage of SNARE-mediated vesicle transport between docking and fusion. During this process, trans-SNARE complexes, formed between molecules in opposing membranes, convert to cis-complexes, with all participants in the same lipid bilayer. Here, we show that glc7 mutant cells accumulate SNARE complexes. These complexes are clearly different from those found in either wild-type or sec18–1 cells as the Sec1p/Munc18 (SM) protein Vps45p does not bind to them. Given that PP1 controls fusion, the SNARE complexes that accumulate in glc7 mutants likely represent trans-SNARE complexes. Vps45p dissociates from the membrane in the absence of PP1 activity, but rapidly reassociates after its reactivation. These data reveal that SM proteins cycle on and off membranes in a stage-specific manner during the vesicle transport reaction, and suggest that protein phosphorylation plays a key role in the regulation of this cycle

Tyrosine phosphorylation of Munc18c on residue 521 abrogates binding to Syntaxin 4

Background: Insulin stimulates exocytosis of GLUT4 from an intracellular store to the cell surface of fat and muscle cells. Fusion of GLUT4-containing vesicles with the plasma membrane requires the SNARE proteins Syntaxin 4, VAMP2 and the regulatory Sec1/Munc18 protein, Munc18c. Syntaxin 4 and Munc18c form a complex that is disrupted upon insulin treatment of adipocytes. Munc18c is tyrosine phosphorylated in response to insulin in these cells. Here, we directly test the hypothesis that tyrosine phosphorylation of Munc18c is responsible for the observed insulin-dependent abrogation of binding between Munc18c and Syntaxin 4. Results: We show that Munc18c is directly phosphorylated by recombinant insulin receptor tyrosine kinase in vitro. Using pull-down assays, we show that phosphorylation abrogates binding of Munc18c to both Syntaxin 4 and the v-SNARE VAMP2, as does the introduction of a phosphomimetic mutation into Munc18c ( Y521E). Conclusion: Our data indicate that insulin-stimulated tyrosine phosphorylation of Munc18c impairs the ability of Munc18c to bind its cognate SNARE proteins, and may therefore represent a regulatory step in GLUT4 traffic

Insulin-stimulated GLUT4 translocation – size is not everything!

Insulin-regulated trafficking of the facilitative glucose transporter GLUT4 has been studied in many cell types. The translocation of GLUT4 from intracellular membranes to the cell surface is often described as a highly specialised form of membrane traffic restricted to certain cell types such as fat and muscle, which are the major storage depots for insulin-stimulated glucose uptake. Here, we discuss evidence that favours the argument that rather than being restricted to specialised cell types, the machinery through which insulin regulates GLUT4 traffic is present in all cell types. This is an important point as it provides confidence in the use of experimentally tractable model systems to interrogate the trafficking itinerary of GLUT4

SNARE phosphorylation: a control mechanism for insulin-stimulated glucose transport and other regulated exocytic events

Trafficking within eukaryotic cells is a complex and highly regulated process; events such as recycling of plasma membrane receptors, formation of multivesicular bodies, regulated release of hormones and delivery of proteins to membranes all require directionality and specificity. The underpinning processes, including cargo selection, membrane fusion, trafficking flow and timing, are controlled by a variety of molecular mechanisms and engage multiple families of lipids and proteins. Here, we will focus on control of trafficking processes via the action of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family of proteins, in particular their regulation by phosphorylation. We will describe how these proteins are controlled in a range of regulated trafficking events, with particular emphasis on the insulin-stimulated delivery of glucose transporters to the surface of adipose and muscle cells. Here, we focus on a few examples of SNARE phosphorylation which exemplify distinct ways in which SNARE machinery phosphorylation may regulate membrane fusion

The Sec1p/Munc18 protein Vps45p binds its cognate SNARE proteins via two distinct modes

Sec1p/Munc18 (SM) proteins are essential for SNARE-mediated membrane trafficking. The formulation of unifying hypotheses for the function of the SM protein family has been hampered by the observation that two of its members bind their cognate syntaxins (Sxs) in strikingly different ways. The SM protein Vps45p binds its Sx Tlg2p in a manner analogous to that captured by the Sly1p–Sed5p crystal structure, whereby the NH2-terminal peptide of the Sx inserts into a hydrophobic pocket on the outer face of domain I of the SM protein. In this study, we report that although this mode of interaction is critical for the binding of Vps45p to Tlg2p, the SM protein also binds Tlg2p-containing SNARE complexes via a second mode that involves neither the NH2 terminus of Tlg2p nor the region of Vps45p that facilitates this interaction. Our findings point to the possibility that SM proteins interact with their cognate SNARE proteins through distinct mechanisms at different stages in the SNARE assembly/disassembly cycle