Reconstitution of SNARE mediated fusion on millisecond timescale

Seminar by Dr. Lukas Tamm, Dept. of Molecular Physiology and Biological Physics, University of Virginia School of Medicine.

Abstract

Despite ten years of knowledge of the structure of the postfusion SNARE complex and the successful reconstitution of SNARE-mediated fusion of proteoliposomes, the mechanism of how SNAREs harness the energy from folding a ternary or quaternary complex to develop a fusion pore is still not known. We have developed new “hybrid” biophysical/structural approaches to address this problem. We have solved by NMR spectroscopy the dynamic structure of full-length membranebound synaptobrevin before it engages with the other partners of the complex. Interestingly, some portions of the SNARE motif are prefolded and others are disordered at this pre-fusion stage, suggesting a new nucleation-stop folding mechanism for SNARE complex formation. In a second approach, to capture fusion intermediates at high time resolution, we have developed a single vesicle fusion assay using total internal reflection fluorescence (TIRF) microscopy in supported bilayers. SNARE-specific fusion of single vesicles commences with a delay of ~18 ms after SNARE docking. A Hodgkin-Huxley inspired analysis of the single vesicle fusion data reveals that each fusion site is composed of 6 to 9 subunits that convert to fusion active species within ~8 ms when the membranes are composed of POPC, DOPS and cholesterol. These kinetically identified subunits most likely represent individual activated SNARE complexes thus revealing the supramolecular architecture of a fusion site. When substantial fractions of DOPE are included into the membranes, the number of required SNARE complexes per fusion site is reduced to 3 to 5, presumably because less energy is required to form curved fusion intermediates in the presence of this cone-shaped lipid. 

References

Ellena, J., Liang, B., Wiktor, M. Cafiso, D.S., Jahn, R., and Tamm, L.K. (2009) Dynamic structure of lipid-bound synaptobrevin suggests a nucleation-propagation mechanism for trans-SNARE complex formation. Proc. Natl. Acad. Sci. USA 106:20306-20311.

Murray, D.H. and Tamm, L.K. (2009) Clustering of syntaxin-1A in model membranes is modulated by phosphatidylinositol-4,5-bisphosphate and cholesterol. Biochemistry 48:4617-4625.

Domanska, M.K. Kiessling, V., Stein, A., Fasshauer, D., and Tamm, L.K. (2009) Single vesicle millisecond fusion kinetics reveals number of SNARE complexes optimal for fast SNARE-mediated membrane fusion. J. Biol. Chem. 284:32158-32166.