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J Biol Chem, Vol. 273, Issue 30, 18784-18792, July 24, 1998
From the 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.
Syndet, an Adipocyte Target SNARE Involved in the Insulin-induced
Translocation of GLUT4 to the Cell Surface
,,,
, and
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
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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