2/14/2011
Kavli Lecture
The Molecular Mechanism of
Intracellular Membrane Fusion
Richard H. Scheller
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The Human Brain
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Neurons Communicate via
Synaptic Transmission
Neuromuscular Synapse
CNS Synapse
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Presynaptic Nerve Terminals Release
Neurotransmitter Stored in Synaptic Vesicles
J.E. Heuser and T.S. Reese
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VAMP1 is a Synaptic Vesicle Protein
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Synaptic Vesicle Proteins in 1990
KD
97.4
66.2
42.7
31.0
W.S. Trimble, … R.H. Scheller (1988)
VAMP1: A synaptic vesicle integral
membrane protein. PNAS 85:4538
T.C. Südhof, … R. Jahn (1989)
A synaptic vesicle membrane protein
(synaptobrevin) is conserved from
mammals to Drosophilia. Neuron 5:1475
21.8
14.4
6
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Vesicle Proteins and Plasma Membrane
Proteins Form a Complex
Synaptotagmin
immunoprecipitation
in three detergents
Syntaxin 
M.K. Bennett, … R.H. Scheller (1992)
Science 257:255
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Syntaxin is Localized to the Plasma Membrane
Syntaxin
M.K. Bennett, … R.H. Scheller (1992) Science 257:255
Synaptophysin
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An Initial
Model of the
Docking and
Fusion Complex
The interaction between p65 (syntaptotagmin) and syntaxin could serve as an intermembrane
scaffold on which the molecular machinery that catalyzes the fusion reaction is assembled. This
fusion machinery is likely to include additional components both of the synaptic vesicle and plasma
membranes as well as soluble factors (29).
M.K. Bennett, … R.H.Scheller (1992) Science 257:255
Reference 29
A Multisubunit Particle Implicated in Membrane Fusion.
“we demonstrate the direct interaction of NSF, SNAPs and an integral membrane component”
D.W. Wilson, … J.E. Rothman (1992) JCB 117:531
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Revised Docking and Fusion Model
Includes SNAP-25
T. Söllner, … J.E. Rothman (1993) Nature 362:318
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Initial Model of the Membrane
Fusion Reaction
T. Söllner, … R.H. Scheller, and J.E. Rothman (1993) Cell 75:409
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FRET Studies Support Parallel Alignment
of Coil Domains
Anti-parallel arrangement
Function: docking vesicles
Syn N –––––––– C*
VAMP C –––––––– N*
Syn N* –––––––– C
VAMP C –––––––– N*
Parallel arrangement
Function: fusing vesicles?
Syn N –––––––– C*
VAMP N* –––––––– C
Syn N* –––––––– C
VAMP N* –––––––– C
trans
cis
R.C. Lin and R.H. Scheller (1997) Neuron 19:1087
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The Mechanism of Membrane Fusion
VAMP
Zippering
Nucleation
SNAP-25
syntaxin
n-sec1
Complete
Membrane
Fusion
n-sec1
SNAP
NSF
ADP+Pi
ATP
R.C. Lin, and R.H. Scheller (1997) Neuron 19:1087
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Cracked PC12 Cell Assay Used to Study
Vesicle Fusion
Developed by T.F.J. Martin
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Docked Vesicles on the Plasma Membrane
of PC12 Cells: A View From the Cytoplasm
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Cracked PC12 Cell Assay Used to Study
Vesicle Fusion
Developed by T.F.J. Martin
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Rescue of BoNT/E Inhibition by
SNAP25 C-terminal Coil
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S25-C Rescues Exocytosis from
BoNT/E-treated PC12 Cells
Y.A. Chen, … R.H. Scheller (1999) Cell 97:165
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16 Layers of Slide Chain Interactions
Mediate SNARE Complex Formation
VAMP
SNAP25
Syntaxin
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Release
(% of wild-type)
The Energy of SNARE Complex
Formation Drives Membrane Fusion
Y.A. Chen, … R.H. Scheller (1999) Cell 97:165
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Syntaxin Q226 Required for
Optimal Efficiency of Dissociation
Sb Arg56
Sn2
Gln174
Sn1
Gln53
Sx
Gln226
S.J. Scales, … R.H. Scheller (2001) PNAS 98:1015
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Members of Vesicle-trafficking Families
S. cerevisiae
C. elegans
D. melanogaster
H. sapiens
SNAREs
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23
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35
Qa-SNAREs/syntaxins
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9
7
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Qb-SNAREs/SNAP Ns
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7
5
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Qc-SNAREs/SNAP Cs
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4
5
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R-SNAREs/VAMPS
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6
5
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Sec1s
4
6
5
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SNARE helical definitions were determined by protein profiling.
Some proteins (SNAP-23, 25, 29) contain two SNARE-coil domains
and thus are counted twice in the coil subdivisions.
J.B. Bock and R.H. Scheller (2001) Nature 409:839
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SNAREs are Strictly Localized to Specific
Cellular Compartments
Golgi
ER
Golgi
Syntaxin510
TGN
Vamp410
VTC
rBet110
Rat pancreas:
NRK
PC12
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Specific Sets of SNARE Proteins Mediate
Intracellular Membrane Trafficking in all Cells
Syntaxin 3,4,5 – M.K. Bennett, …
R.H. Scheller (1993) Cell 74:863
Syntaxin 6 – J.B. Bock, … R.H. Scheller
(1996) JBC 271:17966
GS27, Bet1, SEC22b – J.C. Hay, …
R.H. Scheller (1997) Cell 89:505
Syntaxin 11,13, VAMP 4,7,8 – R.J.
Advani, …R.H. Scheller (1998) JBC
273:10317
Syntaxin 17 – M. Steegmaier, …
R.H. Scheller (2000) Mol. Biol. of the
Cell 11:2719
Many others
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Only Specific SNARE
Combinations are Functional
S.J. Scales, … R.H. Scheller (2000) Neuron 26:457
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The Molecular
Machinery for
Membrane Trafficking
is Conserved from
Yeast to Human Brain
The Molecular Machinery for
secretion is conserved from
yeast to Neurons.
M.K. Bennett and R.H. Scheller
(1993) PNAS 90:2559
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Special Thanks
• Kavli Prize Selection Committee
• Many former members of the Scheller Lab including:
William Trimble
Michael Linial
Mark Bennett
Beverly Wendland
Nicole Calakos
Lisa Elferink
Jonathan Pevsner John Ngsee
Sandy Bajjalieh
Jan Braun
Jason Bock
Raj Advani
Karen Peterson
Tony Ting
Svend Davanger
Martin Steegmaier
Allison Chin
Shu Chan Hsu
Yun Kee
Jesse Hay
Judith Klumperman
William Weiss
Kira Misura
Lino Gonzalez
Richard Lin
Bin Yang
Michael Finley
Rytis Prekeris
Jagath Junutula
David Foletti
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