Membrane Bridging and Hemifusion by Denaturated Munc18

Neuronal Munc18-1 and members of the Sec1/Munc18 (SM) protein family play a critical function(s) in intracellular membrane fusion together with SNARE proteins, but the mechanism of action of SM proteins remains highly enigmatic. During experiments designed to address this question employing a 7-nitrobenz-2-oxa-1,3-diazole (NBD) fluorescence de-quenching assay that is widely used to study lipid mixing between reconstituted proteoliposomes, we observed that Munc18-1 from squid (sMunc18-1) was able to increase the apparent NBD fluorescence emission intensity even in the absence of SNARE proteins. Fluorescence emission scans and dynamic light scattering experiments show that this phenomenon arises at least in part from increased light scattering due to sMunc18-1-induced liposome clustering. Nuclear magnetic resonance and circular dichroism data suggest that, although native sMunc18-1 does not bind significantly to lipids, sMunc18-1 denaturation at 37°C leads to insertion into membranes. The liposome clustering activity of sMunc18-1 can thus be attributed to its ability to bridge two membranes upon (perhaps partial) denaturation; correspondingly, this activity is hindered by addition of glycerol. Cryo-electron microscopy shows that liposome clusters induced by sMunc18-1 include extended interfaces where the bilayers of two liposomes come into very close proximity, and clear hemifusion diaphragms. Although the physiological relevance of our results is uncertain, they emphasize the necessity of complementing fluorescence de-quenching assays with alternative experiments in studies of membrane fusion, as well as the importance of considering the potential effects of protein denaturation. In addition, our data suggest a novel mechanism of membrane hemifusion induced by amphipathic macromolecules that does not involve formation of a stalk intermediate

Liposome clustering activity of sMunc18-1 under different conditions measured by DLS.^a

a<p>DLS measurements of particle size in samples containing protein-free liposomes (POPC∶DOPS 85∶15 molar ratio; 30 µM lipids) and the reagents indicated at the left column. The temperature, incubation time and average radius measured (R_av) are indicated in the other columns.</p

Proposed of model of how a denatured protein can induce membrane hemifusion without proceeding through a stalk intermediate.

<p>The model postulates that denatured proteins (represented as orange randomly shaped curves), and perhaps other amphpathic macromolecules, can induce hemifusion by binding to two membranes (<b>A</b>), accumulating at the membrane-membrane interface (<b>B</b>), and causing a scrambling of lipid molecules at the interface (<b>C</b>) that eventually rearranges into a stable hemifusion diaphragm (<b>D</b>). A curved membrane from a vesicle and a flat membrane are used in the drawings, but the mechanism could apply to membranes with diverse curvatures.</p

sMunc18-1 can induce SNARE-independent increases in the apparent NBD fluorescence intensity in lipid mixing assays.

<p>(<b>A–C</b>) Plots of the ratio between observed fluorescence intensity (F1) and the initial fluorescence intensity (F0) during assays intended to monitor lipid mixing through NBD fluorescence de-quenching. The experiments were performed using proteoliposomes containing synaptobrevin (v) or co-expressed syntaxin-1/SNAP-25 (t) reconstituted using the standard method with a 1∶1000 protein-to-lipid ratio and a lipid composition consisting of POPC∶POPE∶DOPS∶PI∶cholesterol 50∶20∶10∶10∶10 (molar ratio). In the v-SNARE liposomes, 3% of POPC was replaced with 1.5% NBD-PE and 1.5% Rho-PE. In (<b>A</b>), v-SNARE liposomes (50 µM lipids) and t-SNARE liposomes (50 µM lipids) where mixed in the absence of Munc18-1 (black circles), or in the presence of 4 µM rMunc18-1 (red circles) or 4 µM sMunc18-1 (blue circles). In (<b>B</b>), v-SNARE liposomes (50 µM lipids) and t-SNARE liposomes (100 µM lipids) where mixed in the presence of the indicated concentrations of sMunc18-1. In (<b>C</b>), reactions contained v-SNARE liposomes (50 µM lipids) without (black circles) or with 7 µM sMunc18-1 (blue circles), or v-SNARE liposomes (50 µM lipids) and t-SNARE liposomes (100 µM lipids) without (orange circles) or with 7 µM sMunc18-1 (red circles). (<b>D</b>) Lipid mixing assays performed similarly to (A–C) but using protein-free donor liposomes (D) (50 µM lipids) and protein free acceptor liposomes (A) (100 µM lipids) in the absence (black circles) or presence of 7 µM sMunc18-1 (blue circles), or v-SNARE liposomes (50 µM lipids) and t-SNARE liposomes (100 µM lipids) in the absence (orange circles) or presence of 7 µM sMunc18-1 (red circles). For these experiments, the proteoliposomes were prepared with the direct method, using a protein-to-lipid ratio of 1∶1000 and a lipid composition consisting of POPC∶DOPS 85∶15 (molar ratio) (3% of POPC was replaced with 1.5% NBD-PE and 1.5% Rho-PE for donor liposomes and v-SNARE liposomes). All experiments in (<b>A</b>–<b>D</b>) were performed at 37°C monitoring the fluorescence emission intensity at 533 nm (excitation at 460 nm). (<b>E</b>) Fluorescence emission spectra of the sample used to perform the experiments with D+A liposomes and 7 µM sMunc18-1 of panel (<b>D</b>) (blue circles), at the start of the reaction (black trace) and after 1 hr incubation (red trace).</p

Glycerol hinders the liposome clustering activity of sMunc18-1.

<p>(<b>A,B</b>) DLS measurements of particle size in samples containing 15% glycerol, protein-free liposomes (POPC∶DOPS 85∶15 molar ratio; 30 µM lipids) and 4 µM sMunc18-1 right after mixing (<b>A</b>) and after 1 hr incubation at 37°C (<b>B</b>). The average radius (R_av) and polydispersity (Pd) are indicated.</p

Liposome clustering induced by sMunc18-1 is reversed by trypsinolysis.

<p>(<b>A,B</b>) Autocorrelation functions obtained by DLS at different time points after mixing protein-free liposomes (POPC∶DOPS 85∶15 molar ratio; 100 µM lipids) with 7 µM sMunc18-1 (<b>A</b>), and after adding 0.7 µM trypsin at the 20 min time point (<b>B</b>). The insets indicate the color codes for the times at which the data were acquired. Note that the starting point in panel (<b>B</b>) is the same curve as the last point of panel (<b>A</b>), and that the times indicated in panel (<b>B</b>) refer to the beginning of the clustering reaction, rather than the point of trypsin addition. (<b>C</b>) Apparent fluorescence signal intensity at 375 nm (excitation at 350 nm) observed as a function of time after mixing protein-free liposomes (POPC∶DOPS 85∶15 molar ratio; 100 µM lipids) with 7 µM sMunc18-1. Trypsin (0.7 µM) was added to the reaction at 33 min. All the experiments in panels (<b>A–C</b>) were performed at 37°C.</p

Time-dependent binding of sMunc18-1 to lipids.

<p>(<b>A</b>) 1D ¹³C-edited ¹H-NMR spectra of 2 µM ¹³C-labeled sMunc18-1 in the absence or presence of liposomes (POPC∶DOPS 85∶15 molar ratio; 1 mM lipids) at 25°C. (<b>B</b>) 1D ¹³C-edited ¹H-NMR spectra of the same sample containing liposomes in panel (<b>A</b>) acquired as a function of time after raising the temperature to 37°C. (<b>C</b>) 1D ¹³C-edited ¹H-NMR spectra of the same sample lacking liposomes in panel (<b>A</b>) acquired as a function of time after raising the temperature to 37°C.</p