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

doi:10.1074/jbc.273.30.18784

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

From the ^‡Centre for Molecular and Cellular Biology and the Department of Physiology and Pharmacology and the ^¶Centre for Drug Design and Development, University of Queensland, St. Lucia, Queensland, Australia 4072, ^§CSIRO, Division of Molecular Science, 343 Royal Parade, Parkville, Victoria, Australia 3052, and the ^‖Department of Anatomy and Cell Biology, Columbia College of Physicians and Surgeons, New York, New York 10032

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 ∼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.

Footnotes

↵* This work was supported by the National Health and Medical Research Council of Australia. The Center for Molecular and Cellular Biology is a Special Research Center of the Australian Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵** Wellcome Trust Senior Research Fellow. To whom correspondence should be addressed. Tel.: 61 7 3365 4986; Fax: 61 7 3365 4388; E-mail:D.James{at}cmcb.uq.edu.au.
↵1 The abbreviations used are: GLUT4, insulin-regulated glucose transporter; β-OG, β-octylglucoside; GST, glutathione S-transferase; GSVs, GLUT4 storage vesicles; PAGE, polyacrylamide gel electrophoresis; SNAP-25, synaptosomal associated protein of 25 kDa; SNAP-23, SNAP-25-like protein of 23 kDa; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor; SPR, surface plasmon resonance; SSVs, small synaptic vesicles; t-SNARE, target-SNAP receptor; VAMP2, vesicle-associated membrane protein 2; v-SNARE, vesicle-SNAP receptor; FCS, fetal calf serum; PBS, phosphate-buffered saline; PM, plasma membranes; HDM, high density microsomes; LDM, low density microsomes; PVDF, polyvinylidene fluoride; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
↵2 Despite exhibiting greatest sequence similarity to the non-neuronal, SNAP-25-like protein, human SNAP-23 (29), it remains unclear whether Syndet and human SNAP-23 represent the product of the same or two closely related genes (see “Results”).
- Received March 27, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.