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Figure 5.5.
Membrane-directed assembly and the disassembly of SNAREs. Opposing
bilayers containing t-SNARE and v-SNAREs, respectively, interact in a circular array
to form conducting channels in the presence of calcium. (a) Schematic diagram of
the bilayer-electrophysiology setup (EPC9). (b) Lipid vesicle containing nystatin
channels (
red
) and membrane bilayer with SNAREs demonstrate signiicant changes
in capacitance and conductance. When t-SNARE vesicles were added to a v-SNARE
membrane support, the SNAREs in opposing bilayers arranged in a ring pattern,
forming pores as shown in the AFM micrographs (c,d). t-/v-SNARE ring complex
at low (c) and high resolution (d) is shown. Bar = 100 nm. A stepwise increase in
capacitance and conductance (−60 mV holding potential) is demonstrated following
docking and fusion of SNARE-reconstituted vesicles at the SNARE-reconstituted
bilayer of the EPC9 electrophysiological set up (b). Docking and fusion of the vesicle
at the bilayer membrane opens vesicle-associated nystatin channels and SNARE-
induced pore formation, allowing conductance of ions from the
side
of the bilayer membrane (b). Further addition of KCl to induce gradient-driven fusion
resulted in little or no further increase in conductance and capacitance, demonstrating
that docked vesicles have already fused and that the membrane is intact (b). (e-g)
The size of the t-/v-SNARE complex is directly proportional to the size of the SNARE-
reconstituted vesicles. (e) Schematic diagram depicting the interaction of t-SNARE-
reconstituted and v-SNARE-reconstituted liposomes. (f ) AFM images of docked
v-SNARE vesicle at t-SNARE-reconstituted membrane, before and after its dislodge
using the AFM cantilever tip, exposing the t-/v-SNARE-ring complex at the center. (g)
Note the high correlation coeficient between vesicle diameter and size of the SNARE
complex. (h,i) CD data relecting structural changes to SNAREs, both in suspension
and in association with membrane. Structural changes, following the assembly and
disassembly of the t-/v-SNARE complex, are further shown. (h) CD spectra of puriied
full-length SNARE proteins in suspension and (i) in membrane-associated; their
assembly and (NSF-ATP)-induced disassembly is demonstrated. (i) v-SNARE; (ii) t-
SNAREs; (iii) t-/v-SNARE complex; (iv) t-/v-SNARE + NSF; and (v) t-/v-SNARE + NSF +
2.5 mM ATP are shown. CD spectra were recorded at 25 ºC in 5 mM sodium phosphate
buffer (pH 7.5), at a protein concentration of 10 μM. In each experiment, 30 scans were
averaged per sample for enhanced signal to noise, and data were acquired on duplicate
independent samples to ensure reproducibility. (j) Schematic diagram depicting the
possible molecular mechanism of SNARE ring complex formation, when t-SNARE
vesicles and V-SNARE vesicles meet. The process may occur because of a progressive
recruitment of t-/v-SNARE pairs as the opposing vesicles are pulled toward each
other, until a complete ring is established, preventing any further recruitment of t-/v-
SNARE pairs to the complex. The top panel is a side view of two vesicles (one t-SNARE-
reconstituted and the other v-SNARE reconstituted) interacting to form a single t-/v-
SNARE complex, leading progressively (from left to right) to the formation of the ring
complex. The lower panel is a top view of the two interacting vesicles.
cis
to the
trans
30
VAMP and syntaxin are both integral membrane proteins, with the soluble
SNAP-25 associating with syntaxin. Hence, the key to our understanding
of SNARE-induced membrane fusion requires determination of the atomic
arrangement and interaction between membrane-associated v-SNARE and
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