VAMP2, but not VAMP3/cellubrevin, mediates insulin-dependent incorporation of GLUT4 into the plasma membrane of L6 myoblasts.
Like neuronal synaptic vesicles, intracellular GLUT4-containing vesicles must dock and fuse with the plasma membrane, thereby facilitating insulin-regulated glucose uptake into muscle and fat cells. GLUT4 colocalizes in part with the vesicle SNAREs VAMP2 and VAMP3. In this study, we used a single-cell fluorescence-based assay to compare the functional involvement of VAMP2 and VAMP3 in GLUT4 translocation. Transient transfection of proteolytically active tetanus toxin light chain cleaved both VAMP2 and VAMP3 proteins in L6 myoblasts stably expressing exofacially myc-tagged GLUT4 protein and inhibited insulin-stimulated GLUT4 translocation. Tetanus toxin also caused accumulation of the remaining C-terminal VAMP2 and VAMP3 portions in Golgi elements. This behavior was exclusive to these proteins, because the localization of intracellular myc-tagged GLUT4 protein was not affected by the toxin. Upon cotransfection of tetanus toxin with individual vesicle SNARE constructs, only toxin-resistant VAMP2 rescued the inhibition of insulin-dependent GLUT4 translocation by tetanus toxin. Moreover, insulin caused a cortical actin filament reorganization in which GLUT4 and VAMP2, but not VAMP3, were clustered. We propose that VAMP2 is a resident protein of the insulin-sensitive GLUT4 compartment and that the integrity of this protein is required for GLUT4 vesicle incorporation into the cell surface in response to insulin.
Randhawa
V K
VK
Programme in Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
Bilan
P J
PJ
Khayat
Z A
ZA
Daneman
N
N
Liu
Z
Z
Ramlal
T
T
Volchuk
A
A
Peng
X R
XR
Coppola
T
T
Regazzi
R
R
Trimble
W S
WS
Klip
A
A
D000199:Actins / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins; D018482:Muscle, Skeletal / Q000166:cytology; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D050683:R-SNARE Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013744:Tetanus Toxin / Q000378:metabolism; D051040:Vesicle-Associated Membrane Protein 3
D000199:Actins; D051275:Glucose Transporter Type 4; D007328:Insulin; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D016271:Proto-Oncogene Proteins c-myc; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; C495694:Slc2a4 protein, rat; D013744:Tetanus Toxin; D051040:Vesicle-Associated Membrane Protein 3