title: In vitro assay using engineered yeast vacuoles for neuronal SNARE-mediated membrane fusion.
abstract: Intracellular membrane fusion requires not only SNARE proteins but also other regulatory proteins such as the Rab and Sec1/Munc18 (SM) family proteins. Although neuronal SNARE proteins alone can drive the fusion between synthetic liposomes, it remains unclear whether they are also sufficient to induce the fusion of biological membranes. Here, through the use of engineered yeast vacuoles bearing neuronal SNARE proteins, we show that neuronal SNAREs can induce membrane fusion between yeast vacuoles and that this fusion does not require the function of the Rab protein Ypt7p or the SM family protein Vps33p, both of which are essential for normal yeast vacuole fusion. Although excess vacuolar SNARE proteins were also shown to mediate Rab-bypass fusion, this fusion required homotypic fusion and vacuole protein sorting complex, which bears Vps33p and was accompanied by extensive membrane lysis. We also show that this neuronal SNARE-driven vacuole fusion can be stimulated by the neuronal SM protein Munc18 and blocked by botulinum neurotoxin serotype E, a well-known inhibitor of synaptic vesicle fusion. Taken together, our results suggest that neuronal SNARE proteins are sufficient to induce biological membrane fusion, and that this new assay can be used as a simple and complementary method for investigating synaptic vesicle fusion mechanisms.
authors.affiliation: Cell Dynamics Research Center and Bio-imaging Research Center, School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea; and.
authors.forename: Young-Joon
authors.initials: YJ
authors.lastname: Ko
authors.affiliation: Cell Dynamics Research Center and Bio-imaging Research Center, School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea; and.
authors.forename: Miriam
authors.initials: M
authors.lastname: Lee
authors.affiliation: Samsung Biomedical Research Institute and Department of Anatomy and Cell Biology, School of Medicine, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: KyeongJin
authors.initials: K
authors.lastname: Kang
authors.affiliation: Cell Dynamics Research Center and Bio-imaging Research Center, School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea; and.
authors.forename: Woo Keun
authors.initials: WK
authors.lastname: Song
authors.affiliation: Cell Dynamics Research Center and Bio-imaging Research Center, School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea; and junys@gist.ac.kr.
authors.forename: Youngsoo
authors.initials: Y
authors.lastname: Jun
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D001905:Botulinum Toxins; D008561:Membrane Fusion / Q000502:physiology*; D051938:Munc18 Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D016716:PC12 Cells; D051381:Rats; D050600:SNARE Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism
chemical_list: D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; C065454:VPS33 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D001905:Botulinum Toxins; C079005:YPT7 protein, S cerevisiae; D020691:rab GTP-Binding Proteins; C096326:botulinum toxin type E