Syndet, an Adipocyte Target SNARE Involved in the Insulin-induced Translocation of GLUT4 to the Cell Surface

Shane Rea,Laura B Martin,Shane Mcintosh,S Lance Macaulay,Tracie Ramsdale,Giulia Baldini,David E James

doi:10.1074/jbc.273.30.18784

Shane Rea, Laura B Martin + Show 5 more

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https://doi.org/10.1074/jbc.273.30.18784

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Abstract

In adipocytes, insulin stimulates the translocation of the glucose transporter, GLUT4, from an intracellular storage compartment to the cell surface. Substantial evidence exists to suggest that in the basal state GLUT4 resides in discrete storage vesicles. A direct interaction of GLUT4 storage vesicles with the plasma membrane has been implicated because the v-SNARE, vesicle-associated membrane protein-2 (VAMP2), appears to be a specific component of these vesicles. In the present study we sought to identify the cognate target SNAREs for VAMP2 in mouse 3T3-L1 adipocytes. Membrane fractions were isolated from adipocytes and probed by far Western blotting with the cytosolic portion of VAMP2 fused to glutathione S-transferase. Two plasma membrane-enriched proteins, p25 and p35, were specifically labeled with this probe. By using a combination of immunoblotting, detergent extraction, and anion exchange chromatography, we identified p35 as Syntaxin-4 and p25 as the recently identified murine SNAP-25 homologue, Syndet (mSNAP-23). By using surface plasmon resonance we show that VAMP2, Syntaxin-4, and Syndet form a ternary SDS-resistant SNARE complex. Microinjection of anti-Syndet antibodies into 3T3-L1 adipocytes, or incubation of permeabilized adipocytes with a synthetic peptide comprising the C-terminal 24 amino acids of Syndet, inhibited insulin-stimulated GLUT4 translocation to the cell surface by approximately 40%. GLUT1 trafficking remained unaffected by the presence of the peptide. Our data suggest that Syntaxin-4 and Syndet are important cell-surface target SNAREs within adipocytes that regulate docking and fusion of GLUT-4-containing vesicles with the plasma membrane in response to insulin.

Highlights

In adipocytes, insulin stimulates the translocation of the glucose transporter, GLUT4, from an intracellular storage compartment to the cell surface
Based on the above data, we propose that the adipocyte SNAREs Syndet, Syntaxin-4, and vesicle-associated membrane protein-2 (VAMP2) form a stable complex in vivo that constitutes a functional docking intermediate facilitating the specific targeting of GLUT4 vesicles to the cell surface in response to insulin
Substantial evidence suggests that VAMP2 acts as a v-SNARE in the insulin-induced translocation of GLUT4 to the plasma membrane of adipocytes

Summary

Introduction

Insulin stimulates the translocation of the glucose transporter, GLUT4, from an intracellular storage compartment to the cell surface. Our data suggest that Syntaxin-4 and Syndet are important cell-surface target SNAREs within adipocytes that regulate docking and fusion of GLUT-4-containing vesicles with the plasma membrane in response to insulin. The first suggests that under basal conditions, GLUT4 is sequestered within a topologically continuous subdomain of the endosomal system This model assumes that the trafficking of GLUT4 through the general recycling pathway is regulated by its interaction with other proteins that constitute retention factors. One predicted loci of insulin action in this model is the machinery that mediates the docking and fusion of GLUT4 containing vesicles with the plasma membrane Elements of such insulinregulated docking machinery were identified in adipocytes, and some of these molecules were found to be identical to those used for SSV exocytosis in neurons. The discovery of vp165 [12], an aminopeptidase that co-localizes and traffics identically to GLUT4 in response to insulin, provides additional support for the storage vesicle model

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