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Exocytotic fusion pores are composed of both lipids and proteins

Abstract

During exocytosis, fusion pores form the first aqueous connection that allows escape of neurotransmitters and hormones from secretory vesicles. Although it is well established that SNARE proteins catalyze fusion, the structure and composition of fusion pores remain unknown. Here, we exploited the rigid framework and defined size of nanodiscs to interrogate the properties of reconstituted fusion pores, using the neurotransmitter glutamate as a content-mixing marker. Efficient Ca2+-stimulated bilayer fusion, and glutamate release, occurred with approximately two molecules of mouse synaptobrevin 2 reconstituted into 6-nm nanodiscs. The transmembrane domains of SNARE proteins assumed distinct roles in lipid mixing versus content release and were exposed to polar solvent during fusion. Additionally, tryptophan substitutions at specific positions in these transmembrane domains decreased glutamate flux. Together, these findings indicate that the fusion pore is a hybrid structure composed of both lipids and proteins.

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Figure 1: Reconstitution of syb2 into 6- and 13-nm nanodiscs.
Figure 2: Bilayer fusion between v-SNARE nanodiscs (Nd-V) and t-SNARE vesicles.
Figure 3: Reconstitution of glutamate release through nanodiscs.
Figure 4: Distinct structural elements of SNAREs differentially affect bilayer fusion, membrane leakage and glutamate release.
Figure 5: Two molecules of syb2 mediate efficient membrane fusion and content release.
Figure 6: SNARE TMDs are present in the fusion pore.

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Acknowledgements

We thank G. Wagner for providing the MSP1D1ΔH4–H6 plasmid. This study was supported by a grant from the US National Institutes of Health (MH061876 to E.R.C.). H.B. is supported by a postdoctoral fellowship from the Human Frontier Science Program. B.C. and M.G.-O. are supported by funding from the US National Institutes of Health (R01 GM084140 to B.C.). P.J. is supported by Kidney Research UK. J.M.E. is supported by the Biotechnology and Biological Sciences Research Council (BB/J018236/1 to J.M.E.) and Kidney Research UK. E.R.C. is supported as an Investigator of the Howard Hughes Medical Institute.

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Contributions

M.G.-O. performed the single-molecule experiments in the laboratory of B.C. P.J. and J.M.E. carried out the AFM experiments; H.B. performed nanodisc reconstitutions, fusion assays and cysteine-accessibility assays; and H.B. and E.R.C. conceived the project and designed the experiments. H.B. and E.R.C. wrote the paper, and all other authors edited the manuscript.

Corresponding author

Correspondence to Edwin R Chapman.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Characterization of nanodiscs.

(a) Empty nanodiscs were analyzed using a Superdex 200 10/300 GL column equilibrated in 25 mM HEPES pH 7.5, 100 mM KCl, 1mM DTT and 5% glycerol. (b) AFM images of v-SNARE nanodiscs. (c) t-SNARE nanodiscs were analyzed using a Superdex 200 10/300 column as described in panel A. (d) Lipid mixing between t -SNARE nanodiscs and v-SNARE vesicles was monitored in the presence of Ca2+ (1 mM) and C2AB, with and without cd-v.

Supplementary Figure 2 Fusion pores close after fusion.

(a) Illustration of lipid mixing and dithionite quenching experiments. (b-c) Protection of NBD fluorescence is not observed using empty nanodiscs (b) or when dithionite is added at the beginning of the fusion reaction (c). (d) Left Panel: illustration of the co-flotation assay. Samples containing syb2 nanodiscs and t-SNARE liposomes were mixed with an equal volume of 80% Accudenz, and then layered with 30% and 0% Accudenze. Ultra-centrifugation was carried out at 55,000 rpm at 4 °C for 2 h. Samples were collected at the 40% Accudenze layer and at the 30%-0% interface. Right panel: DY650-labeled syb2 nanodiscs (Nd-V) were incubated with or without t-SNARE liposomes (T-liposome) at 37 °C for 1 h in the presence of Ca2+ (1 mM) and C2AB (1 μM). Samples were analyzed by co-flotation, and the top and bottom fractions were subjected to SDS-PAGE and fluorescence scanning. (e) Syb2 nanodiscs were incubated with t-SNARE liposomes at 37 °C for 1 h, followed by AFM analysis. Arrows indicate free nanodiscs; arrowheads indicate liposome-associated nanodiscs. Scale bar: 100 nm.

Supplementary Figure 3 Glutamate release during SUV-nanodisc fusion as a function of 6-nm Nd-V concentration.

Data were fitted to the Hill (black dotted line) and Michaelis-Menten equations (red line). Data points are presented as means ± s.d. from three independent trials.

Supplementary Figure 4 Reconstitution of Ca2+-triggered lipid mixing and glutamate release with full-length Syt1.

(a) Lipid mixing between t-SNARE vesicles and nanodiscs harboring syb2 and full-length syt1. (b) Glutamate release from t-SNARE vesicles in the presence and absence of nanodiscs harboring syb2 and full-length syt1. Arrow indicates the addition of Ca2+ (1 mM final concentration).

Supplementary Figure 5 Efficient Ca2+-triggered bilayer fusion and glutamate release occurs with two v-SNAREs in 6-nm nanodiscs.

(a) Left panel: SDS-PAGE of nanodiscs (ND1, ND2, ND3, ND4, ND5, ND6, ND7 and ND8). Right panel: densitometry of the syb2 protein bands from the gel in panel (a) were plotted versus the syb2 copy number per nanodisc. (b) Size exclusion chromatography of 6-nm ND2. (c) Lipid mixing between 6-nm ND2 and t-SNARE vesicles, in the presence and absence of C2AB; addition of Ca2+ (1 mM final concentration) is indicated by an arrow. (d) Glutamate release from t-SNARE vesicles in the presence of 6-nm ND2 and C2AB; the arrow indicates the addition of Ca2+ (1 mM final concentration). (e) Experimental setup for imaging bleaching steps from arrays of zero-mode waveguide nanoholes containing single nanodiscs that bear fluorescently labeled syb2. (f) Illustration of an individual zero-mode waveguide nanohole with a nanodisc containing two syb2-DY650 molecules immobilized on the glass surface via biotin/streptavidin. Fluorescence from DY650 was measured upon epi laser illumination, with the effective excitation volume limited to attoliters. (g) Brightfield image of an array of zero-mode waveguide nanoholes with diameters of ~ 200 nm. (h) Fluorescence image of an array as in (g) after sparse labeling with syb2-DY650 containing nanodiscs such that only a few nanoholes contained a single nanodisc (one of these is indicated with a yellow circle).

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Bao, H., Goldschen-Ohm, M., Jeggle, P. et al. Exocytotic fusion pores are composed of both lipids and proteins. Nat Struct Mol Biol 23, 67–73 (2016). https://doi.org/10.1038/nsmb.3141

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