Supplementary Materials Supplemental Materials supp_24_8_1176__index. the synaptic factor Munc18-1 (Shen is the protein concentration (mM), is the measured ellipticity (mdeg), is the number of residues, and is the path BIRB-796 inhibition length (cm). Electron microscopy EM imaging of liposomes was carried out at the Boulder Laboratory for 3-D Electron Microscopy of Cells (Boulder, CO). Brain lipid extract (Folch fraction I; Sigma-Aldrich, St. Louis, MO) was dried and resuspended at 1 mg/ml in HEPES-buffered saline (50 mM HEPES, pH 7.4, and 100 mM NaCl). The resuspended lipid bilayers were then sonicated for 10 min to generate Folch liposomes. Folch liposomes (0.3 mg/ml) were incubated with WT or mutant Doc2b proteins (10 M) in the presence of BIRB-796 inhibition 1 mM EDTA or CaCl2 for 10 h at room temperature. Subsequently the samples were stained with 2% uranyl acetate and observed under a Philips CM100 transmission electron microscope (Philips, Amsterdam, Netherlands). Supplementary Material Supplemental Materials: Click here to view. Acknowledgments We thank Tom Giddings (University of Colorado at Boulder) for assistance with electron microscopy, Brooke Hirsch (University of Colorado School of Medicine, Aurora, CO) for assistance with circular dichroism measurements, and Thomas Sdhof (Stanford University, Stanford, CA) for providing plasmids encoding Doc2b mutants defective in Ca2+ binding. This work was supported by National Institutes of Health Pathway to Independence Award “type”:”entrez-nucleotide”,”attrs”:”text”:”DK080080″,”term_id”:”187587700″,”term_text”:”DK080080″DK080080 and National Institutes of Health Grant DK095367 to J.S. J.S. is a Pew Scholar in the Biomedical Sciences. Abbreviations used: Doc2double C2-like domainsFRETF?rster (fluorescence) resonance energy transferSNAREsoluble em N /em -ethylmaleimideCsensitive factor attachment protein receptorVAMPvesicle-associated membrane proteinWTwide type Footnotes This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E12-11-0810) on February 20, 2013. REFERENCES Bhalla A, Tucker WC, Chapman ER. Synaptotagmin isoforms couple distinct ranges of Ca2+, Ba2+, and Sr2+ concentration to SNARE-mediated membrane fusion. Mol Biol Cell. 2005;16:4755C4764. [PMC free article] [PubMed] [Google Scholar]Birnbaum MJ. Identification of a novel gene encoding an insulin-responsive glucose transporter protein. Cell. 1989;57:305C315. [PubMed] [Google Scholar]Blot V, McGraw TE. Molecular mechanisms controlling GLUT4 intracellular retention. Mol Biol Cell. 2008;19:3477C3487. [PMC free article] [PubMed] [Google Scholar]Brandie FM, Aran V, Verma A, McNew JA, Bryant NJ, Gould GW. Negative regulation of syntaxin4/SNAP-23/VAMP2-mediated membrane fusion by Munc18c in vitro. PLoS One. 2008;3:e4074. [PMC free article] [PubMed] [Google Scholar]Brozinick JT, Jr, Reynolds TH, Dean D, Cartee G, Cushman SW. 1-[N, O-bis-(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine (KN-62), an inhibitor of calcium-dependent camodulin protein kinase II, inhibits both insulin- and hypoxia-stimulated glucose transport in skeletal muscle. Biochem J. 1999;339:533C540. [PMC free article] [PubMed] [Google Scholar]Bryant NJ, Govers R, James DE. Regulated transport of the glucose transporter GLUT4. Nat Rev Mol Cell Biol. 2002;3:267C277. [PubMed] [Google Scholar]Burgoyne RD, Morgan A. Membrane trafficking: three steps to fusion. Curr Biol. 2007;17:R255CR258. [PubMed] [Google Scholar]Chapman ER. How does synaptotagmin trigger neurotransmitter release. Annu Rev Biochem. 2008;77:615C641. [PubMed] [Google Scholar]Charron MJ, Brosius FC, 3rd, Alper SL, Lodish HF. A glucose transport protein expressed predominately in insulin-responsive tissues. Proc Natl Acad Sci USA. 1989;86:2535C2539. [PMC free article] [PubMed] [Google Scholar]Chernomordik LV, Kozlov MM. Membrane hemifusion: crossing a chasm in two leaps. Cell. 2005;123:375C382. [PubMed] [Google Scholar]Chou MM, Hou W, Johnson J, Graham LK, Lee MH, Chen CS, Newton AC, Schaffhausen BS, Toker A. Regulation of protein kinase C zeta PIK3CD by PI 3-kinase and PDK-1. Curr Biol. 1998;8:1069C1077. [PubMed] [Google Scholar]D’Andrea-Merrins M, Chang L, Lam AD, Ernst SA, Stuenkel EL. Munc18c interaction with syntaxin 4 monomers and SNARE complex intermediates in GLUT4 vesicle trafficking. J Biol Chem. 2007;282:16553C16566. [PubMed] [Google Scholar]Fernandez I, Arac D, Ubach J, Gerber SH, Shin O, Gao Y, Anderson RG, Sudhof BIRB-796 inhibition TC, Rizo J. Three-dimensional structure of the synaptotagmin 1 C2B-domain: synaptotagmin 1 as a phospholipid binding machine. Neuron. 2001;32:1057C1069. [PubMed] [Google Scholar]Fukuda.