Membrane bridging and hemifusion by denaturated Munc18

Area of Science:

• Molecular Biology
• Biophysics
• Cell Biology

Background:

• Sec1/Munc18 (SM) proteins are crucial for intracellular membrane fusion with SNARE proteins.
• The precise mechanism of SM protein action in membrane fusion remains poorly understood.
• Fluorescence de-quenching assays are commonly used to study lipid mixing in membrane fusion.

Purpose of the Study:

• To investigate the mechanism of action of neuronal Munc18-1 in membrane fusion.
• To elucidate the role of squid Munc18-1 (sMunc18-1) in membrane fusion processes.
• To address the enigmatic function of SM proteins in intracellular membrane fusion.

Main Methods:

• Utilized a 7-nitrobenz-2-oxa-1,3-diazole (NBD) fluorescence de-quenching assay.
• Performed fluorescence emission scans and dynamic light scattering experiments.
• Employed nuclear magnetic resonance, circular dichroism, and cryo-electron microscopy.

Main Results:

• sMunc18-1 increased NBD fluorescence intensity and induced liposome clustering, even without SNARE proteins.
• Denaturation of sMunc18-1 at 37 °C led to membrane insertion and liposome bridging.
• Cryo-electron microscopy revealed hemifusion diaphragms in sMunc18-1-induced liposome clusters.

Conclusions:

• sMunc18-1-induced liposome clustering and hemifusion arise from bridging membranes post-denaturation.
• Findings underscore the importance of complementary assays and considering protein denaturation in membrane fusion studies.
• A novel mechanism for membrane hemifusion mediated by amphipathic macromolecules, independent of stalk intermediates, is suggested.