Speaker: Mary Munson, University of Massachusetts Medical School
Title: Activation of the Yeast Exocyst,Tethering Complex for SNARE Regulation and Membrane Fusion
Abstract: A major challenge for a molecular understanding of membrane trafficking has been the elucidation of high resolution structures of large, multi-subunit tethering complexes that spatially and temporally control intracellular membrane fusion. Exocyst is a hetero-octameric protein complex, proposed to tether secretory vesicles at the plasma membrane, and to provide quality control of SNARE-mediated membrane fusion. Breakthroughs in methodologies, including sample preparation, biochemical characterization, fluorescence and single-particle cryo-EM, are providing critical insights into the structure and function of the exocyst. We are investigating how the yeast exocyst interacts with SNARE proteins to control SNARE complex formation and membrane fusion. Intriguingly, fully assembled exocyst interacts weakly with the individual SNAREs, SNARE complexes, and the SNARE regulator Sec1, despite previously observed robust SNARE binding with recombinant proteins. Using an auxin-inducible degradation system and mutant yeast strains, we purified exocyst subcomplexes and mutant complexes and showed that several have increased affinities for the different SNAREs. Negative stain EM was used to visualize the structure of an exocyst subcomplex, as well as the 3D structure of an activated mutant exocyst complex. Comparison of the negative stain images to the cryo-EM structure of fully assembled exocyst revealed that several subunits become more dynamic and accessible for SNARE interactions. We propose that exocyst needs to become activated and undergo a conformational change, in order to efficiently interact with and regulate the SNAREs for membrane fusion.
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