Hemifusion in SNARE-mediated membrane fusion

SNAREs are essential for intracellular membrane fusion. Using EPR, we determined the structure of the transmembrane domain (TMD) of the vesicle (v)-SNARE Snc2p involved in trafficking in yeast. Structural features of the TMD were used to design a v-SNARE mutant in which about half of the TMD was deleted. Liposomes containing this mutant induced outer leaflet mixing but not inner leaflet mixing when incubated with liposomes containing target membrane (t)-SNAREs. Hemifusion was also detected with wild-type SNAREs when low protein concentrations were reconstituted. Thus, these results show that SNARE-mediated fusion can transit through a hemifusion intermediate.

[1]  A. Brünger,et al.  Analysis of a Yeast SNARE Complex Reveals Remarkable Similarity to the Neuronal SNARE Complex and a Novel Function for the C Terminus of the SNAP-25 Homolog, Sec9* , 1997, The Journal of Biological Chemistry.

[2]  Lin Yang,et al.  Observation of a Membrane Fusion Intermediate Structure , 2002, Science.

[3]  H. Khorana,et al.  A collision gradient method to determine the immersion depth of nitroxides in lipid bilayers: application to spin-labeled mutants of bacteriorhodopsin. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[4]  M. Kozlov,et al.  Protein-lipid interplay in fusion and fission of biological membranes. , 2003, Annual review of biochemistry.

[5]  L. Tamm.  Hypothesis: spring-loaded boomerang mechanism of influenza hemagglutinin-mediated membrane fusion. , 2003, Biochimica et biophysica acta.

[6]  Y. Shin,et al.  The membrane topology of the fusion peptide region of influenza hemagglutinin determined by spin-labeling EPR. , 1997, Journal of molecular biology.

[7]  J. White,et al.  The Many Mechanisms of Viral Membrane Fusion Proteins , 2005, Current topics in microbiology and immunology.

[8]  Matthias Mann,et al.  Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion , 2001, Nature.

[9]  A. Janoff,et al.  Novel inner monolayer fusion assays reveal differential monolayer mixing associated with cation-dependent membrane fusion. , 2000, Biochimica et biophysica acta.

[10]  J. Rothman,et al.  Compartmental specificity of cellular membrane fusion encoded in SNARE proteins , 2000, Nature.

[11]  Y. Shin,et al.  Insertion of the Membrane-proximal Region of the Neuronal SNARE Coiled Coil into the Membrane* , 2003, The Journal of Biological Chemistry.

[12]  Y. Shin,et al.  Probing domain swapping for the neuronal SNARE complex with electron paramagnetic resonance. , 2002, Biochemistry.

[13]  Reinhard Jahn,et al.  Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution , 1998, Nature.

[14]  W. Xiao,et al.  The synaptic SNARE complex is a parallel four-stranded helical bundle , 1998, Nature Structural Biology.

[15]  Steven S. Vogel,et al.  Lysolipids reversibly inhibit Ca2+‐, GTP‐ and pH‐dependent fusion of biological membranes , 1993, FEBS letters.

[16]  M. Roth,et al.  A point mutation in the transmembrane domain of the hemagglutinin of influenza virus stabilizes a hemifusion intermediate that can transit to fusion. , 2000, Molecular biology of the cell.

[17]  Y. Shin,et al.  The membrane-dipped neuronal SNARE complex: a site-directed spin labeling electron paramagnetic resonance study. , 2002, Biochemistry.

[18]  P. Bronk,et al.  The Pathway of Membrane Fusion Catalyzed by Influenza Hemagglutinin: Restriction of Lipids, Hemifusion, and Lipidic Fusion Pore Formation , 1998, The Journal of cell biology.

[19]  Thorsten Lang,et al.  Membrane fusion. , 2002, Current opinion in cell biology.

[20]  Y. Shin,et al.  Constitutive versus regulated SNARE assembly: a structural basis , 2004, The EMBO journal.

[21]  W. Hubbell,et al.  Recent advances in site-directed spin labeling of proteins. , 1998, Current opinion in structural biology.

[22]  K. Hideg,et al.  Motion of spin-labeled side chains in T4 lysozyme. Correlation with protein structure and dynamics. , 1996, Biochemistry.

[23]  L. Chernomordik,et al.  Kinetically differentiating influenza hemagglutinin fusion and hemifusion machines. , 2003, Biophysical journal.

[24]  Y. Shin,et al.  HIV-1 gp41 tertiary structure studied by EPR spectroscopy. , 1996, Biochemistry.

[25]  Misuzu Baba,et al.  Geranylgeranylated Snares Are Dominant Inhibitors of Membrane Fusion , 2000, The Journal of cell biology.

[26]  R. T. Armstrong,et al.  The Transmembrane Domain of Influenza Hemagglutinin Exhibits a Stringent Length Requirement to Support the Hemifusion to Fusion Transition , 2000, The Journal of cell biology.

[27]  Sejal M. Patel,et al.  SNARE Complex Formation Is Triggered by Ca2+ and Drives Membrane Fusion , 1999, Cell.

[28]  Benedikt Westermann,et al.  SNAREpins: Minimal Machinery for Membrane Fusion , 1998, Cell.

[29]  J. Rothman,et al.  Close Is Not Enough , 2000, The Journal of cell biology.

[30]  J. Rothman,et al.  Mechanisms of intracellular protein transport , 1994, Nature.

[31]  J. Skehel,et al.  Coiled Coils in Both Intracellular Vesicle and Viral Membrane Fusion , 1998, Cell.

[32]  Dae-Hyuk Kweon,et al.  Regulation of neuronal SNARE assembly by the membrane , 2003, Nature Structural Biology.

[33]  Judith M. White,et al.  Lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion , 1994, Cell.

[34]  P. S. Kim,et al.  HIV Entry and Its Inhibition , 1998, Cell.

[35]  M. Jackson,et al.  Transmembrane Segments of Syntaxin Line the Fusion Pore of Ca2+-Triggered Exocytosis , 2004, Science.