N-ethylmaleimide-sensitive fusion protein (NSF) and its co-factor soluble NSF attachment protein (alpha)-SNAP) are essential components of the synaptic vesicle fusion machinery and form part of a structurally-conserved 20S protein complex. However, their precise function, relative to fusion itself, is not clear. Using a UV-activated cross-linking approach, we have measured the rate at which a single round of NSF-driven ATP hydrolysis leads to 20S complex disassembly within synaptic membranes. Although this rate is substantially faster than previous estimates of NSF-dependent ATP hydrolysis, it remains much lower than published rates for fusion of synaptic vesicles. Furthermore, the stability of 20S complexes is unaffected by Ca(2+) at concentrations that elicit rapid membrane fusion. We conclude that the ATPase activity of NSF does not contribute directly to vesicle fusion, but more likely plays an earlier role in the synaptic vesicle cycle.

REFERENCES

Barnard
R. O.
,
Morgan
A.
,
Burgoyne
R. D.
(
1997
).
Stimulation of NSF ATPase activity by-SNAP is required for SNARE complex disassembly and exocytosis.
J. Cell Biol
139
,
875
883
Baumert
M.
,
Maycox
P. R.
,
Navone
F.
,
De Camilli
P.
,
Jahn
R.
(
1989
).
Synaptobrevin: an integral membrane protein of 18,000 daltons present in small synaptic vesicles of rat brain.
EMBO J
8
,
379
384
Bennett
M. K.
,
Calakos
N.
,
Scheller
R. H.
(
1992
).
Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones.
Science
257
,
255
259
Binz
T.
,
Blasi
J.
,
Yamasaki
S.
,
Baumeister
A.
,
Link
E.
,
Sudhof
T. C.
,
Jahn
R.
,
Niemann
H.
(
1994
).
Proteolysis of SNAP-25 by types E and A botulinal neurotoxins.
J. Biol. Chem
269
,
1617
1620
Bruns
D.
,
Jahn
R.
(
1995
).
Real-time measurement of transmitter release from single synaptic vesicles.
Nature
377
,
62
65
Burgoyne
R. D.
,
Morgan
A.
(
1998
).
Analysis of regulated exocytosis in adrenal chromaffin cells: insights into NSF/SNAP/SNARE function.
BioEssays
20
,
328
335
Chen
Y. A.
,
Scales
S. J.
,
Patel
S. M.
,
Doung
Y.-C.
,
Scheller
R. H.
(
1999
).
SNARE complex formation is triggered by Ca2+and drives membrane fusion.
Cell
97
,
165
174
Clary
D. O.
,
Griff
I. C.
,
Rothman
J. E.
(
1990
).
SNAPs, a family of NSF attachment proteins involved in intracellular membrane fusion in animals and yeast.
Cell
61
,
709
721
Coorsen
J. R.
,
Blank
P. S.
,
Tahara
M.
,
Zimmerberg
J.
(
1998
).
Biochemical and functional studies of cortical vesicle fusion: the SNARE complex and Ca++sensitivity.
J. Cell Biol
143
,
1845
1857
Fasshauer
D.
,
Otto
H.
,
Eliason
W. K.
,
Jahn
R.
,
Brunger
A. T.
(
1997
).
Structural changes are associated with soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex formation.
J. Biol. Chem
272
,
28036
28041
Fasshauer
D.
,
Sutton
R. B.
,
Brunger
A. T.
,
Jahn
R.
(
1998
).
Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q-and R-SNAREs.
Proc. Nat. Acad. Sci. USA
95
,
15781
15786
Görlich
D.
,
Kurzchalia
T. V.
,
Wiedmann
M.
,
Rapoport
T. A.
(
1991
).
Probing the molecular environment of translocating polypeptide chains by cross-linking.
Meth. Cell Biol
34
,
241
262
Hanson
P. I.
,
Roth
R.
,
Morisaki
R.
,
Jahn
R.
,
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
536
Hay
J. C.
,
Scheller
R. H.
(
1997
).
SNAREs and NSF in targeted membrane fusion.
Curr. Opin. Cell Biol
9
,
505
512
Hayashi
T.
,
McMahon
H.
,
Yamasaki
S.
,
Binz
T.
,
Hata
Y.
,
Sudhof
T. C.
,
Niemann
H.
(
1994
).
Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly.
EMBO J
13
,
5051
5061
Hayashi
T.
,
Yamasaki
S.
,
Nauenburg
S.
,
Binz
T.
,
Niemann
H.
(
1995
).
Disassembly of the reconstituted synaptic vesicle membrane fusion complex in vitro.
EMBO J
14
,
2317
2325
Haynes
L. P.
,
Barnard
R. J. O.
,
Morgan
A.
,
Burgoyne
R. D.
(
1998
).
Stimulation of NSF ATPase activity during t-SNARE priming.
FEBS Lett
436
,
1
5
Heidelberger
R.
,
Heinemann
C.
,
Neher
E.
,
Matthews
G.
(
1994
).
Calcium dependence of the rate of exocytosis in a synaptic terminal.
Nature
371
,
513
515
Hohl
T. M.
,
Parlati
F.
,
Wimmer
C.
,
Rothman
J. E.
,
Söllner
T. H.
,
Engelhardt
H.
(
1998
).
Arrangement of subunits in 20S particles consisting of NSF, SNAPs, and SNARE complexes.
Mol. Cell
2
,
539
548
Hughson
F. M.
(
1999
).
Membrane fusion: structure snared at last.
Curr. Biol
9
,
49
–.
Krieg
U. C.
,
Walter
P.
,
Johnson
A. E.
(
1986
).
Photocrosslinking of the signal sequence of nascent preprolactin to the 54-kilodalton polypeptide of the signal recognition particle.
Proc. Nat. Acad. Sci. USA
83
,
8604
8608
Mayer
A.
,
Wickner
W.
,
Haas
A.
(
1996
).
SEC18p (NSF)- driven release of SEC17p (-SNAP) can precede docking and fusion of yeast vacuoles.
Cell
85
,
83
94
Mehta
P. P.
,
Battenberg
E.
,
Wilson
M. C.
(
1996
).
SNAP-25 andsynaptotagmin involvement in the final Ca2+-dependent triggering of neurotransmitter exocytosis.
Proc. Nat. Acad. Sci. USA
93
,
10471
10476
Ninomiya
Y.
,
Kishimoto
T.
,
Yamazawa
T.
,
Ikeda
H.
,
Miyashita
Y.
,
Kasai
H.
(
1997
).
Kinetic diversity in the fusion of exocytotic vesicles.
EMBO J
16
,
929
934
Novick
P.
,
Zerial
M.
(
1997
).
The diversity of Rab proteins in vesicle transport.
Curr. Opin. Cell Biol
9
,
496
504
O'Connor
V.
,
Augustine
G. J.
,
Betz
H.
(
1994
).
Synaptic vesicle exocytosis: molecules and models.
Cell
76
,
785
787
Otter-Nilsson
M.
,
Hendriks
R.
,
Pecheur-Huet
E.-I.
,
Hoekstra
D.
,
Nilsson
T.
(
1999
).
Cytosolic ATPases, p97 and NSF, are sufficient to mediate rapid membrane fusion.
EMBO J
18
,
2074
2083
Otto
H.
,
Hanson
P. I.
,
Jahn
R.
(
1997
).
Assembly and disassembly ofa ternary complex of synaptobrevin, syntaxin, and SNAP-25 in the membrane of synaptic vesicles.
Proc. Nat. Acad. Sci. USA
94
,
6197
6120
Oyler
G. A.
,
Higgins
G. A.
,
Hart
R. A.
,
Battenberg
E.
,
Billingsley
M.
,
Bloom
F. E.
,
Wilson
M. C.
(
1989
).
The identification of a novel synaptosomal-associated protein, SNAP-25, differentially expressed by neuronal subpopulations.
J. Cell Biol
109
,
3039
3052
Pause
A.
,
Sonenberg
N.
(
1992
).
Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A.
EMBO J
11
,
2643
2654
Pellegrini
L. L.
,
O'Connor
V.
,
Betz
H.
(
1994
).
Fusion complex formation protects synaptobrevin against proteolysis by tetanus toxin light chain.
FEBS Lett
353
,
319
323
Peters
C.
,
Mayer
A.
(
1998
).
Ca2+/calmodulin signals the completion of docking and triggers a late step of vacuole fusion.
Nature
396
,
575
580
Poirier
M. A.
,
Xiao
W.
,
Macosko
J. C.
,
Chan
C.
,
Shin
Y.-K.
,
Bennett
M. K.
(
1998
).
The synaptic SNARE complex is a parallel four-stranded helical bundle.
Nature Struct. Biol
5
,
765
769
Rothman
J. E.
(
1994
).
Mechanism of intracellular protein transport.
Nature
372
,
55
63
Rothman
J. E.
,
Warren
G.
(
1994
).
Implications of the SNARE hypothesis for intracellular membrane topology and dynamics.
Curr. Biol
4
,
220
223
Schiavo
G.
,
Stenbeck
G.
,
Rothman
J. E.
,
Söllner
T. H.
) (
1997
).
Binding of the synaptic vesicle v-SNARE, synaptotagmin, to the plasmamembrane t-SNARE, SNAP-25, can explain docked vesicles at neurotoxin-treated synapses.
Proc. Nat. Acad. Sci. USA
94
,
997
1001
Schoenmakers
T. J. M.
,
Visser
G. J.
,
Flik
G.
,
Theuvenet
A. P. R.
(
1992
).
CHELATOR: an improved method for computing metal ion concentrations in physiological solutions.
BioTechniques
12
,
870
879
Schweizer
F. E.
,
Dresbach
T.
,
DeBello
W. M.
,
O'Connor
V.
,
Augustine
G. J.
,
Betz
H.
(
1998
).
Regulation of neurotransmitter release kinetics by NSF.
Science
279
,
1203
1206
Skehel
J. J.
,
Wiley
D. C.
(
1998
).
Coiled coils in both intracellular and viral membrane fusion.
Cell
95
,
871
874
Söllner
T.
,
Bennett
M. K.
,
Whiteheart
S. W.
,
Scheller
R. H.
,
Rothman
J. E.
(
1993
).
A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion.
Cell
75
,
409
418
Söllner
T.
,
Whiteheart
S. W.
,
Brunner
M.
,
Erdjument-Bromage
H.
,
Geromanos
S.
,
Tempst
P.
,
Rothman
J. E.
(
1993
).
SNAP receptors implicated in vesicle targeting and fusion.
Nature
362
,
318
324
Swanton
E.
,
Sheehan
J.
,
Bishop
N.
,
High
S.
,
Woodman
P.
(
1998
).
Formation and turnover of NSF-and SNAP-containing “fusion” complexes occur on undocked, clathrin-coated vesicle-derived membranes.
Mol. Biol. Cell
9
,
1633
1647
Tahara
M.
,
Coorsen
J. R.
,
Timmers
K.
,
Blank
P. S.
,
Whalley
T.
,
Scheller
R.
,
Zimmerberg
J.
(
1998
).
Calcium can disrupt the SNARE protein complex on sea urchin egg secretory vesicles without irreversibly blocking fusion.
J. Biol. Chem
273
,
33667
33673
Trimble
W. S.
,
Cowan
D. M.
,
Scheller
R. H.
(
1988
).
VAMP-1: A synaptic vesicle-associated integral membrane protein.
Proc. Nat. Acad. Sci. USA
85
,
4538
4542
Ungermann
C.
,
Sato
K.
,
Wickner
W.
(
1998
).
Defining the functions of trans-SNARE pairs.
Nature
396
,
543
548
Weber
T.
,
Zemelman
B. V.
,
McNew
J. A.
,
Westermann
B.
,
Gmachl
M.
,
Parlati
F.
,
Söllner
T. H.
,
Rothman
J. E.
(
1998
).
SNAREpins: minimal machinery for membrane fusion.
Cell
92
,
759
772
Xu
T.
,
Ashery
U.
,
Burgoyne
R. D.
,
Neher
E.
(
1999
).
Early requirement for-SNAP and NSF in the secretory cascade in chromaffin cells.
EMBO J
18
,
3293
3304
This content is only available via PDF.