Energy-dependent disassembly of self-assembled SNARE complex: observation at nanometer resolution using atomic force...

Area of Science:

• Biochemistry
• Cell Biology
• Membrane Biophysics

Background:

• Soluble NSF Attachment Protein Receptors (SNAREs) mediate membrane fusion.
• The precise structural requirements for SNARE-mediated pore formation are not fully understood.

Purpose of the Study:

• To investigate the role of membrane association in t-/v-SNARE complex formation and pore generation.
• To determine the conditions necessary for the formation of functional, conducting pores.

Main Methods:

• Reconstitution of full-length v-SNARE proteins into lipid vesicles.
• Reconstitution of t-SNARE proteins into lipid membranes.
• Co-incubation of v-SNARE vesicles with t-SNARE membranes.
• Observation of complex assembly and pore formation using biophysical techniques.
• Disassembly assays using NSF-ATP.

Main Results:

• Membrane-associated v-SNAREs and t-SNAREs self-assemble into ring-like structures, forming pores and bridging opposing bilayers.
• v-SNARE proteins alone, without liposomes, form t-/v-SNARE complexes but lack the ring structure and do not form pores or bilayer continuity.
• Functional pore formation requires membrane association of both t-SNAREs and v-SNAREs in a circular array, dependent on calcium.
• NSF-ATP effectively disassembles both membrane-associated and non-membrane-associated t-/v-SNARE complexes.

Conclusions:

• Membrane association is critical for the formation of functional, ring-like t-/v-SNARE complexes that create conducting pores.
• The NSF-ATP system provides a mechanism for regulating SNARE complex disassembly, irrespective of their structural arrangement.