title: Doc2b promotes GLUT4 exocytosis by activating the SNARE-mediated fusion reaction in a calcium- and membrane bending-dependent manner.
abstract: The glucose transporter GLUT4 plays a central role in maintaining body glucose homeostasis. On insulin stimulation, GLUT4-containing vesicles fuse with the plasma membrane, relocating GLUT4 from intracellular reservoirs to the cell surface to uptake excess blood glucose. The GLUT4 vesicle fusion reaction requires soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) as the core fusion engine and a group of regulatory proteins. In particular, the soluble C2-domain factor Doc2b plays a key role in GLUT4 vesicle fusion, but its molecular mechanism has been unclear. Here we reconstituted the SNARE-dependent GLUT4 vesicle fusion in a defined proteoliposome fusion system. We observed that Doc2b binds to GLUT4 exocytic SNAREs and potently accelerates the fusion kinetics in the presence of Ca(2+). The stimulatory activity of Doc2b requires intact Ca(2+)-binding sites on both the C2A and C2B domains. Using electron microscopy, we observed that Doc2b strongly bends the membrane bilayer, and this membrane-bending activity is essential to the stimulatory function of Doc2b in fusion. These results demonstrate that Doc2b promotes GLUT4 exocytosis by accelerating the SNARE-dependent fusion reaction by a Ca(2+)- and membrane bending-dependent mechanism. Of importance, certain features of Doc2b function appear to be distinct from how synaptotagmin-1 promotes synaptic neurotransmitter release, suggesting that exocytic Ca(2+) sensors may possess divergent mechanisms in regulating vesicle fusion.
authors: Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309, USA.
authors: Haijia
authors: H
authors: Yu
authors: Shailendra S
authors: SS
authors: Rathore
authors: Eric M
authors: EM
authors: Davis
authors: Yan
authors: Y
authors: Ouyang
authors: Jingshi
authors: J
authors: Shen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry*; D021961:Cell Membrane Structures / Q000737:chemistry; D005089:Exocytosis; D051275:Glucose Transporter Type 4 / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D008081:Liposomes / Q000737:chemistry / Q000648:ultrastructure; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D050600:SNARE Proteins / Q000737:chemistry*
chemical_list: D002135:Calcium-Binding Proteins; C490422:Doc2b protein, rat; D051275:Glucose Transporter Type 4; D008081:Liposomes; D009419:Nerve Tissue Proteins; D050600:SNARE Proteins; C495694:Slc2a4 protein, rat; D002118:Calcium