Abstract
The exocyst is an octameric protein complex implicated in tethering secretory vesicles to the plasma membrane during exocytosis. To provide a mechanistic understanding of how it functions, it is of critical importance to elucidate its three-dimensional structure. This chapter briefly describes the protocols used in our structure determination of Exo70p and Exo84p, two subunits of the exocyst from Saccharomyces cerevisiae. Folding and domain arrangements of both proteins are predicted using bioinformatics tools. Limited proteolysis is carried out to define the boundaries of folded structures, which guides the design of suitable constructs for protein crystallization. The solved structures of both proteins validate the strategy and suggest it might be also used for structural studies of other proteins alike.
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Acknowledgements
The original crystallization and structure determination were carried out in the group of Karin M. Reinisch at the Yale School of Medicine. Proteolysis analysis was repeated using the same protocols to generate Figs. 4a and 5a. The experiments were carried out in our group at the Max F. Perutz Laboratories in the Medical University of Vienna under the support of grant P24383-B21 from the Austrian Science Fund (FWF) to GD.
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Lesigang, J., Dong, G. (2016). Analysis of Three-Dimensional Structures of Exocyst Components. In: Sanchez-Diaz, A., Perez, P. (eds) Yeast Cytokinesis. Methods in Molecular Biology, vol 1369. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3145-3_14
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DOI: https://doi.org/10.1007/978-1-4939-3145-3_14
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