Synaptic Transmission
Mike Edwardson
Introduction
At the cellular level, membrane fusion is essential for transport of large molecules in and
out of the cell and within the cell between organelles. At the physiological level, membrane
fusion controls many important bodily processes, including vesicular release of
neurotransmitters at the synapse for nervous transmission and hormonal release from
endocrine glands. This significant phenomenon has been investigated for almost half a
century, however, most has been achieved in the recent 15-20 years, culminating in
identification of membrane fusion molecular machinery complex.
George Palade, awarded a Nobel Prize in Physiology and Medicine in 1974, was one of the
first scientists who proposed that vesicular system connects organelles within the cell,
however, at the time lacked the tools to investigate how spatiotemporal specificity of
vesicular transport is achieved. An accepted hypothesis was the fusion takes place after
recognition of protein-based “barcodes”. The very beginning of the journey to discover
these “barcodes” was marked by seminal experiments by Schekman and Rothman who
conducted genetic screens in yeast and biochemical cell free assay respectively. These and
other following experiments as well as structural data from X-ray crystallography and EM
revealed that vesicle fusion is driven by membrane proteins with large extracellular
domains, called SNAREs, that “zipped” together. As SNAREs would be present on both of the
membranes, this allowed to bring the membranes into proximity for fusion. Other SNARE-
binding proteins, including complexins, tethering factors and more have been discovered
and their roles in mediating membrane fusion and formation of SNARE complex are still
being elucidated.
Reading List 2018-19
Lecture 1. Identification of components of the membrane fusion machinery
Original papers
Novick, P., Field, C. and Schekman, R. (1980) Identification of 23 complementation groups
required for post-translational events in the yeast secretory pathway. Cell 21, 205-215
Balch, W.E., Dunphy, W.G., Braell, W.A. and Rothman, J.E. (1984) Reconstitution of the
transport of protein between successive compartments of the Golgi measured by the
coupled incorporation of N-acetylglucosamine. Cell 39, 405-416
Malhotra, V., Orci, L., Glick, B.S., Block, M.R. and Rothman, J.E. (1988) Role of an N-
ethylmaleimide-sensitive transport component in promoting fusion of transport vesicles
with cisternae of the Golgi stack. Cell 54, 221-227
Clary, D.O., Griff, I.C. and Rothman, J.E. (1990) SNAPs, a family of NSF attachment proteins
involved in intracellular membrane fusion in animals and yeast. Cell 61, 709-721
Söllner, T., Whiteheart, S.W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst,
P. and Rothman, J.E. (1993) SNAP receptors implicated in vesicle targeting and fusion.
Nature 362, 318-324
,Hanson, P.I., Roth, R, Morisaki, H., Jahn, R. and Heuser, J.E. (1997) Structure and
conformational changes in NSF and its membrane receptor complexes visualized by
quick-freeze/deep-etch electron microscopy. Cell 90, 523-535
Sutton, R.B., Fasshauer, D., Jahn, R. and Brunger, A. (1998) Crystal structure of a SNARE
complex involved in synaptic exocytosis at 2.4 Å resolution. Nature 395, 347-353
Reviews
Palade, G.E. (1975) Intracellular aspects of protein synthesis. Science 189, 347-358 (of
historical interest)
Brunger, A.T. (2001) Structure of proteins involved in synaptic vesicle fusion in neurons.
Annu. Rev. Biophys. Biomol. Struct. 30, 157-171
,Lecture 2. SNAREs and membrane fusion
Original papers
Schiavo, G., Benfenati, F., Poulain, B., Rossetto, O., Polverino de Laureto, P., DasGupta, B.R.
and Montecucco, C. (1992) Tetanus and botulinum-B neurotoxins block neurotransmitter
release by proteolytic cleavage of synaptobrevin. Nature 359, 832-835
Chai, Q., Arndt, J.W., Dong, M., Tepp, W.H., Johnson, E.A., Chapman, E.R. and Stevens R.C.
(2006) Structural basis of cell surface receptor recognition by botulinum neurotoxin B.
Nature. 444, 1096-1100
Weber, T., Zemelman, B.V., McNew, J.A., Parlati, F., Söllner, T.H. and Rothman, J.E. (1998)
SNAREpins: minimal machinery for membrane fusion. Cell 92, 759-772
Takamori, S., et al. (2006) Molecular anatomy of a trafficking organelle. Cell 127, 831-846
Dennison, S.M., Bowen, M.E., Brunger, A.T. and Lentz, B.R. (2006) Neuronal SNAREs do not
trigger fusion between synthetic membranes but do promote PEG-mediated membrane
fusion. Biophys. J. 90, 1661-1675
Zhao, M, Wu, S., Zhou, Q., Vivona, S., Cipriano, D.J., Cheng, Y. and Brunger, A.T. (2015)
Mechanistic insights into the recycling machine of the SNARE complex. Nature 518, 61-67
Mayer, A., Wickner, W. and Haas, A. (1996) Sec18p (NSF)-driven release of Sec17p ( -SNAP)
can precede docking and fusion of yeast vacuoles. Cell 85, 83-94
McNew, J.A., Parlati, F., Fukuda, R., Johnston, R.J., Paz, K., Paumet, F., Söllner, T.H. and
Rothman, J.E. (2000) Compartmental specificity of cellular membrane fusion encoded in
SNARE proteins. Nature 407, 153-159
Wong, M. and Munro, S. (2014) Membrane trafficking. The specificity of vesicle traffic to the
Golgi is encoded in the golgin coiled-coil proteins. Science 346, 1256898
Peters, C., Bayer, M.J., Bühler, S., Andersen, J.S., Mann, M. and Mayer, A. (2001) Trans-
complex formation by proteolipid channels in the terminal phase of membrane fusion.
Nature 409, 581-588
Strasser, B., Iwaszkiewicz, J., Michielin, O. and Mayer, A. (2011) The V-ATPase proteolipid
cylinder promotes the lipid-mixing stage of SNARE-dependent fusion of yeast vacuoles.
EMBO J. 30, 4126-4141
Reviews
Jahn, R. and Scheller, R.H. (2006) SNAREs – engines for membrane fusion. Nat. Rev. Mol. Cell
Biol. 7, 631-643
Martens, S. and McMahon, H.T. (2008) Mechanisms of membrane fusion: disparate players
and common principles. Nat. Rev. Mol. Cell Biol. 9, 543-556
Montal, M. (2010) Botulinum neurotoxin: a marvel of protein design. Annu Rev Biochem. 79,
591-617
, Lecture 3. Ca2+ sensing at the nerve terminal
Original papers
Perin, M.S., Fried, V.A., Mignery, G.A., Jahn, R. and Südhof, T.C. (1990) Phospholipid binding
by a synaptic vesicle protein homologous to the regulatory region of protein kinase C.
Nature 345, 260- 263
Chapman, E.R., Hanson, P.I., An, S. and Jahn, R. (1995) Ca2+ regulates the interaction
between synaptotagmin and syntaxin. J. Biol. Chem. 270, 23667-23671
Bai, J., Tucker, W.C. and Chapman, E.R. (2004) PIP2 increases the speed of response of
synaptotagmin and steers its membrane-penetration activity toward the plasma
membrane. Nat. Struct. Mol. Biol. 1, 36-44
Chapman, E.R. and Davis, A.F. (1998) Direct interaction of a Ca2+-binding loop of
synaptotagmin with lipid bilayers. J. Biol. Chem. 273, 13995-14001.
Davis, A.F., Bai, J., Fasshauer, D., Wolowick, M.J., Lewis, J.L. and Chapman, E.R. (1999)
Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex
onto membranes. Neuron 24, 363-376
Liu, H., Bai, H., Xue, R., Takahashi, H., Edwardson, J.M. and Chapman, E.R. (2014) Linker
mutations dissociate the function of synaptotagmin I during evoked and spontaneous
release and reveal membrane penetration as a step during excitation-secretion coupling.
Nat. Neurosci. 17, 670-677
Tucker, W.C., Weber, T. and Chapman, E.R. (2004) Reconstitution of Ca2+-regulated
membrane fusion by synaptotagmin and SNAREs. Science 304, 435-438
Stein, A., Radhakrishnan, A., Riedel, D., Fasshauer, D. and Jahn, R. (2007) Synaptotagmin
activates membrane fusion through a Ca2+-dependent trans interaction with
phospholipids. Nat. Struct. Mol. Biol. 14, 904-911
Hui, E., et al (2009) Synaptotagmin-mediated bending of the target membrane is a critical
step in Ca2+-regulated fusion. Cell 138, 709-721
Yao, J., Gaffaney, J.D., Kwon, S.E. and Chapman, E.R. (2011) Doc2 is a Ca2+ sensor required
for asynchronous neurotransmitter release. Cell 147, 666-677
Reviews
Chapman, E.R. (2008) How does synaptotagmin trigger neurotransmitter release? Annu.
Rev. Biochem. 77, 615-641
McMahon, H.T., Kozlov, M.M. and Martens, S. (2010) Membrane curvature in synaptic
vesicle fusion and beyond. Cell 140, 601-605
Südhof, T.C. (2013) Neurotransmitter release: the last millisecond in the life of a synaptic
vesicle. Neuron 80, 675-690
Mike Edwardson
Introduction
At the cellular level, membrane fusion is essential for transport of large molecules in and
out of the cell and within the cell between organelles. At the physiological level, membrane
fusion controls many important bodily processes, including vesicular release of
neurotransmitters at the synapse for nervous transmission and hormonal release from
endocrine glands. This significant phenomenon has been investigated for almost half a
century, however, most has been achieved in the recent 15-20 years, culminating in
identification of membrane fusion molecular machinery complex.
George Palade, awarded a Nobel Prize in Physiology and Medicine in 1974, was one of the
first scientists who proposed that vesicular system connects organelles within the cell,
however, at the time lacked the tools to investigate how spatiotemporal specificity of
vesicular transport is achieved. An accepted hypothesis was the fusion takes place after
recognition of protein-based “barcodes”. The very beginning of the journey to discover
these “barcodes” was marked by seminal experiments by Schekman and Rothman who
conducted genetic screens in yeast and biochemical cell free assay respectively. These and
other following experiments as well as structural data from X-ray crystallography and EM
revealed that vesicle fusion is driven by membrane proteins with large extracellular
domains, called SNAREs, that “zipped” together. As SNAREs would be present on both of the
membranes, this allowed to bring the membranes into proximity for fusion. Other SNARE-
binding proteins, including complexins, tethering factors and more have been discovered
and their roles in mediating membrane fusion and formation of SNARE complex are still
being elucidated.
Reading List 2018-19
Lecture 1. Identification of components of the membrane fusion machinery
Original papers
Novick, P., Field, C. and Schekman, R. (1980) Identification of 23 complementation groups
required for post-translational events in the yeast secretory pathway. Cell 21, 205-215
Balch, W.E., Dunphy, W.G., Braell, W.A. and Rothman, J.E. (1984) Reconstitution of the
transport of protein between successive compartments of the Golgi measured by the
coupled incorporation of N-acetylglucosamine. Cell 39, 405-416
Malhotra, V., Orci, L., Glick, B.S., Block, M.R. and Rothman, J.E. (1988) Role of an N-
ethylmaleimide-sensitive transport component in promoting fusion of transport vesicles
with cisternae of the Golgi stack. Cell 54, 221-227
Clary, D.O., Griff, I.C. and Rothman, J.E. (1990) SNAPs, a family of NSF attachment proteins
involved in intracellular membrane fusion in animals and yeast. Cell 61, 709-721
Söllner, T., Whiteheart, S.W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst,
P. and Rothman, J.E. (1993) SNAP receptors implicated in vesicle targeting and fusion.
Nature 362, 318-324
,Hanson, P.I., Roth, R, Morisaki, H., Jahn, R. and Heuser, J.E. (1997) Structure and
conformational changes in NSF and its membrane receptor complexes visualized by
quick-freeze/deep-etch electron microscopy. Cell 90, 523-535
Sutton, R.B., Fasshauer, D., Jahn, R. and Brunger, A. (1998) Crystal structure of a SNARE
complex involved in synaptic exocytosis at 2.4 Å resolution. Nature 395, 347-353
Reviews
Palade, G.E. (1975) Intracellular aspects of protein synthesis. Science 189, 347-358 (of
historical interest)
Brunger, A.T. (2001) Structure of proteins involved in synaptic vesicle fusion in neurons.
Annu. Rev. Biophys. Biomol. Struct. 30, 157-171
,Lecture 2. SNAREs and membrane fusion
Original papers
Schiavo, G., Benfenati, F., Poulain, B., Rossetto, O., Polverino de Laureto, P., DasGupta, B.R.
and Montecucco, C. (1992) Tetanus and botulinum-B neurotoxins block neurotransmitter
release by proteolytic cleavage of synaptobrevin. Nature 359, 832-835
Chai, Q., Arndt, J.W., Dong, M., Tepp, W.H., Johnson, E.A., Chapman, E.R. and Stevens R.C.
(2006) Structural basis of cell surface receptor recognition by botulinum neurotoxin B.
Nature. 444, 1096-1100
Weber, T., Zemelman, B.V., McNew, J.A., Parlati, F., Söllner, T.H. and Rothman, J.E. (1998)
SNAREpins: minimal machinery for membrane fusion. Cell 92, 759-772
Takamori, S., et al. (2006) Molecular anatomy of a trafficking organelle. Cell 127, 831-846
Dennison, S.M., Bowen, M.E., Brunger, A.T. and Lentz, B.R. (2006) Neuronal SNAREs do not
trigger fusion between synthetic membranes but do promote PEG-mediated membrane
fusion. Biophys. J. 90, 1661-1675
Zhao, M, Wu, S., Zhou, Q., Vivona, S., Cipriano, D.J., Cheng, Y. and Brunger, A.T. (2015)
Mechanistic insights into the recycling machine of the SNARE complex. Nature 518, 61-67
Mayer, A., Wickner, W. and Haas, A. (1996) Sec18p (NSF)-driven release of Sec17p ( -SNAP)
can precede docking and fusion of yeast vacuoles. Cell 85, 83-94
McNew, J.A., Parlati, F., Fukuda, R., Johnston, R.J., Paz, K., Paumet, F., Söllner, T.H. and
Rothman, J.E. (2000) Compartmental specificity of cellular membrane fusion encoded in
SNARE proteins. Nature 407, 153-159
Wong, M. and Munro, S. (2014) Membrane trafficking. The specificity of vesicle traffic to the
Golgi is encoded in the golgin coiled-coil proteins. Science 346, 1256898
Peters, C., Bayer, M.J., Bühler, S., Andersen, J.S., Mann, M. and Mayer, A. (2001) Trans-
complex formation by proteolipid channels in the terminal phase of membrane fusion.
Nature 409, 581-588
Strasser, B., Iwaszkiewicz, J., Michielin, O. and Mayer, A. (2011) The V-ATPase proteolipid
cylinder promotes the lipid-mixing stage of SNARE-dependent fusion of yeast vacuoles.
EMBO J. 30, 4126-4141
Reviews
Jahn, R. and Scheller, R.H. (2006) SNAREs – engines for membrane fusion. Nat. Rev. Mol. Cell
Biol. 7, 631-643
Martens, S. and McMahon, H.T. (2008) Mechanisms of membrane fusion: disparate players
and common principles. Nat. Rev. Mol. Cell Biol. 9, 543-556
Montal, M. (2010) Botulinum neurotoxin: a marvel of protein design. Annu Rev Biochem. 79,
591-617
, Lecture 3. Ca2+ sensing at the nerve terminal
Original papers
Perin, M.S., Fried, V.A., Mignery, G.A., Jahn, R. and Südhof, T.C. (1990) Phospholipid binding
by a synaptic vesicle protein homologous to the regulatory region of protein kinase C.
Nature 345, 260- 263
Chapman, E.R., Hanson, P.I., An, S. and Jahn, R. (1995) Ca2+ regulates the interaction
between synaptotagmin and syntaxin. J. Biol. Chem. 270, 23667-23671
Bai, J., Tucker, W.C. and Chapman, E.R. (2004) PIP2 increases the speed of response of
synaptotagmin and steers its membrane-penetration activity toward the plasma
membrane. Nat. Struct. Mol. Biol. 1, 36-44
Chapman, E.R. and Davis, A.F. (1998) Direct interaction of a Ca2+-binding loop of
synaptotagmin with lipid bilayers. J. Biol. Chem. 273, 13995-14001.
Davis, A.F., Bai, J., Fasshauer, D., Wolowick, M.J., Lewis, J.L. and Chapman, E.R. (1999)
Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex
onto membranes. Neuron 24, 363-376
Liu, H., Bai, H., Xue, R., Takahashi, H., Edwardson, J.M. and Chapman, E.R. (2014) Linker
mutations dissociate the function of synaptotagmin I during evoked and spontaneous
release and reveal membrane penetration as a step during excitation-secretion coupling.
Nat. Neurosci. 17, 670-677
Tucker, W.C., Weber, T. and Chapman, E.R. (2004) Reconstitution of Ca2+-regulated
membrane fusion by synaptotagmin and SNAREs. Science 304, 435-438
Stein, A., Radhakrishnan, A., Riedel, D., Fasshauer, D. and Jahn, R. (2007) Synaptotagmin
activates membrane fusion through a Ca2+-dependent trans interaction with
phospholipids. Nat. Struct. Mol. Biol. 14, 904-911
Hui, E., et al (2009) Synaptotagmin-mediated bending of the target membrane is a critical
step in Ca2+-regulated fusion. Cell 138, 709-721
Yao, J., Gaffaney, J.D., Kwon, S.E. and Chapman, E.R. (2011) Doc2 is a Ca2+ sensor required
for asynchronous neurotransmitter release. Cell 147, 666-677
Reviews
Chapman, E.R. (2008) How does synaptotagmin trigger neurotransmitter release? Annu.
Rev. Biochem. 77, 615-641
McMahon, H.T., Kozlov, M.M. and Martens, S. (2010) Membrane curvature in synaptic
vesicle fusion and beyond. Cell 140, 601-605
Südhof, T.C. (2013) Neurotransmitter release: the last millisecond in the life of a synaptic
vesicle. Neuron 80, 675-690
