Doc2b promotes GLUT4 exocytosis by activating the SNARE-mediated fusion reaction in a calcium- and membrane bending-dependent manner

• Published in: Molecular biology of the cell Vol. 24; no. 8; pp. 1176 - 1184
• Main Authors: Yu, Haijia, Rathore, Shailendra S, Davis, Eric M, Ouyang, Yan, Shen, Jingshi
• Format: Journal Article
• Language: English
• Published: United States The American Society for Cell Biology 15.04.2013
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Calcium - chemistry; Calcium-Binding Proteins - chemistry; Cell Membrane - chemistry; Cell Membrane Structures - chemistry; Exocytosis; Glucose Transporter Type 4 - chemistry; Hydrophobic and Hydrophilic Interactions; Liposomes - chemistry; Liposomes - ultrastructure; Models, Molecular; Nerve Tissue Proteins - chemistry; Protein Binding; Protein Structure, Tertiary; Rats; SNARE Proteins - chemistry
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.e12-11-0810

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.