Membrane-directed molecular assembly of the neuronal SNARE complex.
Bottom Line: Using high-resolution electron microscopy, the electron density maps and 3D topography of the membrane-directed SNARE ring complex was determined at nanometre resolution.Furthermore, the mathematical prediction of the SNARE ring complex size with reasonable accuracy, and the possible mechanism of membrane-directed t-/v-SNARE ring complex assembly, was determined from the study.Therefore in the present study, using both lipososome-reconstituted recombinant t-/v-SNARE proteins, and native v-SNARE present in isolated SV membrane, the membrane-directed molecular assembly of the neuronal SNARE complex was determined for the first time and its size mathematically predicted.
Affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA.Show MeSH
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Mentions: Because majority of intracellular fusion events occur between vesicular compartments, it was imperative to determine the structure and arrangement of the t-/v-SNARE complex when t-SNARE-vesicles and v-SNARE-vesicles meet. In this case, because both opposing membrane would exhibit curvature, we expected the v- and t-SNAREs to interact at a point, attracting additional SNARE pairs to form a t-/v-SNARE bundle. To test our hypothesis, two sets of 50 nm diameter liposomes were reconstituted with t-SNAREs and v-SNARE respectively (Fig. 1A–C), for use in our study. Surprisingly, exposure of 50 nm v-SNARE-liposome to 50 nm t-SNARE-liposome resulted in the establishment of a highly stable approximately 8 nm diameter t-/v-SNARE ring complex. Once formed, the t-/v-SNARE complex is highly stable, resistant even to a membrane solubilizing anionic detergent, sodium dodecyl sulphate. Therefore, following membrane solubilization (Fig. 1B, D), the stable SNARE complexes  have enabled a detailed examination of its morphology at high resolution, using both AFM and electron microscopy (EM) in the current study.
Affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA.