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Concurrent binding of complexin and synaptotagmin to liposome-embedded SNARE complexes.

Chicka MC, Chapman ER - Biochemistry (2009)

Bottom Line: Synaptotagmin and complexin regulate SNARE-mediated synaptic vesicle exocytosis.It has been proposed that complexin clamps membrane fusion and that Ca(2+)-synaptotagmin displaces complexin from SNARE complexes to relieve this clamping activity.Moreover, the clamping ability of apo-synaptotagmin occluded the clamping activity of complexin until the arrival of a Ca(2+) trigger, at which point synaptotagmin accelerated fusion while high concentrations of complexin inhibited fusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Programs in Cellular and Molecular Biology, University of Wisconsin, 1300 University Avenue, SMI 129, Madison, Wisconsin 53706, USA.

ABSTRACT
Synaptotagmin and complexin regulate SNARE-mediated synaptic vesicle exocytosis. It has been proposed that complexin clamps membrane fusion and that Ca(2+)-synaptotagmin displaces complexin from SNARE complexes to relieve this clamping activity. Using a reconstituted system, we demonstrate that complexin and synaptotagmin simultaneously bind to neuronal SNARE complexes and that both apo-synaptotagmin and complexin inhibit SNARE-mediated membrane fusion. Moreover, the clamping ability of apo-synaptotagmin occluded the clamping activity of complexin until the arrival of a Ca(2+) trigger, at which point synaptotagmin accelerated fusion while high concentrations of complexin inhibited fusion. Thus, the inhibitory patterns of synaptotagmin and complexin are different, suggesting that SNAREs assemble into distinct states along the fusion pathway. These data also suggest that during synaptotagmin-regulated vesicle-vesicle fusion, complexin does not function as a fusion clamp that is relieved by Ca(2+)-synaptotagmin.

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Binding of cpx-I and syt to membrane-embedded ternary SNARE complexes. (a) Diagram of the flotation assay used to monitor binding interactions (see also Figure S1 of the Supporting Information). (b) Cpx-I (10 μM) and increasing concentrations of syt were added to ternary SNARE complexes individually or together and then the complexes subjected to flotation. Vesicles harbored 70% PC and 30% PE. Binding was monitored in the absence or presence of Ca2+ (1 mM), and proteins were visualized by being stained with Coomassie blue. (c) Experiments in panel b were repeated using vesicles harboring PS (55% PC, 30% PE, and 15% PS). All gels are representative from n ≥ 3.
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fig1: Binding of cpx-I and syt to membrane-embedded ternary SNARE complexes. (a) Diagram of the flotation assay used to monitor binding interactions (see also Figure S1 of the Supporting Information). (b) Cpx-I (10 μM) and increasing concentrations of syt were added to ternary SNARE complexes individually or together and then the complexes subjected to flotation. Vesicles harbored 70% PC and 30% PE. Binding was monitored in the absence or presence of Ca2+ (1 mM), and proteins were visualized by being stained with Coomassie blue. (c) Experiments in panel b were repeated using vesicles harboring PS (55% PC, 30% PE, and 15% PS). All gels are representative from n ≥ 3.

Mentions: Next, we addressed whether syt and cpx-I simultaneously bind SNAREs and whether cpx-I-mediated inhibition can be overcome by Ca2+-syt (as proposed in refs (4)−(6) and (8)). We titrated syt onto ternary SNARE complexes that had been saturated with cpx-I (Figure 1a,b) in the coflotation assay. For these experiments, we used liposomes that lacked phosphatidylserine (PS). t-SNARE vesicles lacking PS must be used for this experiment, in contrast to ref (6), because Ca2+-syt binds with high affinity to PS (16). With the omission of PS, any coflotation of syt with the vesicles must be solely due to direct interactions between syt and SNARE proteins. Even when present in a 3-fold molar excess of cpx-I, syt had no effect on the extent of cpx-I binding either in the absence or in the presence of Ca2+ (Figure 1b). Similarly, saturation of ternary SNARE complexes with cpx-I did not affect the extent of syt binding (Figure 1b). These data unambiguously demonstrate that syt and complexin simultaneously bind SNARE complexes that have been assembled onto liposomes [similar results were obtained with cpx-I(26−83) (Figure S3 of the Supporting Information)].


Concurrent binding of complexin and synaptotagmin to liposome-embedded SNARE complexes.

Chicka MC, Chapman ER - Biochemistry (2009)

Binding of cpx-I and syt to membrane-embedded ternary SNARE complexes. (a) Diagram of the flotation assay used to monitor binding interactions (see also Figure S1 of the Supporting Information). (b) Cpx-I (10 μM) and increasing concentrations of syt were added to ternary SNARE complexes individually or together and then the complexes subjected to flotation. Vesicles harbored 70% PC and 30% PE. Binding was monitored in the absence or presence of Ca2+ (1 mM), and proteins were visualized by being stained with Coomassie blue. (c) Experiments in panel b were repeated using vesicles harboring PS (55% PC, 30% PE, and 15% PS). All gels are representative from n ≥ 3.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651691&req=5

fig1: Binding of cpx-I and syt to membrane-embedded ternary SNARE complexes. (a) Diagram of the flotation assay used to monitor binding interactions (see also Figure S1 of the Supporting Information). (b) Cpx-I (10 μM) and increasing concentrations of syt were added to ternary SNARE complexes individually or together and then the complexes subjected to flotation. Vesicles harbored 70% PC and 30% PE. Binding was monitored in the absence or presence of Ca2+ (1 mM), and proteins were visualized by being stained with Coomassie blue. (c) Experiments in panel b were repeated using vesicles harboring PS (55% PC, 30% PE, and 15% PS). All gels are representative from n ≥ 3.
Mentions: Next, we addressed whether syt and cpx-I simultaneously bind SNAREs and whether cpx-I-mediated inhibition can be overcome by Ca2+-syt (as proposed in refs (4)−(6) and (8)). We titrated syt onto ternary SNARE complexes that had been saturated with cpx-I (Figure 1a,b) in the coflotation assay. For these experiments, we used liposomes that lacked phosphatidylserine (PS). t-SNARE vesicles lacking PS must be used for this experiment, in contrast to ref (6), because Ca2+-syt binds with high affinity to PS (16). With the omission of PS, any coflotation of syt with the vesicles must be solely due to direct interactions between syt and SNARE proteins. Even when present in a 3-fold molar excess of cpx-I, syt had no effect on the extent of cpx-I binding either in the absence or in the presence of Ca2+ (Figure 1b). Similarly, saturation of ternary SNARE complexes with cpx-I did not affect the extent of syt binding (Figure 1b). These data unambiguously demonstrate that syt and complexin simultaneously bind SNARE complexes that have been assembled onto liposomes [similar results were obtained with cpx-I(26−83) (Figure S3 of the Supporting Information)].

Bottom Line: Synaptotagmin and complexin regulate SNARE-mediated synaptic vesicle exocytosis.It has been proposed that complexin clamps membrane fusion and that Ca(2+)-synaptotagmin displaces complexin from SNARE complexes to relieve this clamping activity.Moreover, the clamping ability of apo-synaptotagmin occluded the clamping activity of complexin until the arrival of a Ca(2+) trigger, at which point synaptotagmin accelerated fusion while high concentrations of complexin inhibited fusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Programs in Cellular and Molecular Biology, University of Wisconsin, 1300 University Avenue, SMI 129, Madison, Wisconsin 53706, USA.

ABSTRACT
Synaptotagmin and complexin regulate SNARE-mediated synaptic vesicle exocytosis. It has been proposed that complexin clamps membrane fusion and that Ca(2+)-synaptotagmin displaces complexin from SNARE complexes to relieve this clamping activity. Using a reconstituted system, we demonstrate that complexin and synaptotagmin simultaneously bind to neuronal SNARE complexes and that both apo-synaptotagmin and complexin inhibit SNARE-mediated membrane fusion. Moreover, the clamping ability of apo-synaptotagmin occluded the clamping activity of complexin until the arrival of a Ca(2+) trigger, at which point synaptotagmin accelerated fusion while high concentrations of complexin inhibited fusion. Thus, the inhibitory patterns of synaptotagmin and complexin are different, suggesting that SNAREs assemble into distinct states along the fusion pathway. These data also suggest that during synaptotagmin-regulated vesicle-vesicle fusion, complexin does not function as a fusion clamp that is relieved by Ca(2+)-synaptotagmin.

Show MeSH
Related in: MedlinePlus