A functional role for VAP-33 in insulin-stimulated GLUT4 traffic

Soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNA REs) are critical proteins in membrane fusion, in both regulated and consti tutive vesicular traffic. In addition, proteins that interact with the SNAR Es are thought to regulate fusion. Vesicle-associated membrane protein-2 (V AMP-2) is a SNARE protein involved in insulin-dependent glucose transporter 4 (GLUT4) traffic. VAMP-2 is required for productive GLUT4 incorporation i nto the plasma membrane. VAMP-associated protein of 33 kDa (VAP-33) is an i ntegral membrane protein that binds VAMPs in vitro, and is hypothesized to be a regulator of VAMPs. In L6 skeletal myoblasts, which display insulin-de pendent traffic of GLUT4. we show that VAP-33 colocalized significantly wit h VAMP-2 using indirect confocal immunofluorescence and biochemical cosegre gation. Overexpression of wild-type VAP-33 in L6 myoblasts attenuated the i nsulin-dependent incorporation of myc-tagged GLUT4 into the plasma membrane , and this response was restored by co-overexpression of VAMP-2 linked to g reen fluorescent protein. Antibodies to VAP-33 microinjected into 3T3-L1 ad ipocytes abrogated the insulin-stimulated translocation of GLUT4 to the pla sma membrane, as measured in adhered plasma membrane lawns. Immunopurified VAMP-2-containing compartments from L6 myotubes and 3T3-L1 adipocytes showe d significant levels of VAP-33. We propose that VAP-33 may be a regulator o f VAMP-2 availability for GLUT4 traffic and other vesicle fusion events.