| Description |
The exocyst is a conserved octameric protein complex in eukaryotes that is essential for tethering and fusion of vesicles to the plasma membrane. Composed of eight subunits, it is essential for growth, secretion, and developmental processes and has been implicated in cell motility, autophagy, cell division, and exocytosis. Functional defects are linked to cancer, neurodegeneration, ciliopathies, and bacterial pathogenesis. With known biochemical data from interacting partners and four crystal structures known, architectural models have proposed extended, rod-like conformations with side-by-side packing. Isolation and physical characterization of this essential protein complex has remained elusive. Structural information is necessary to determine how this protein functions in the context of exocytosis. Using the Saccharomyces cerevisiae model eukaryotic organism, this research aims to purify intact exocyst complex, characterize it structurally, and identify the spatial orientation of the carboxyl termini of Sec15 relative to the fully assembled complex. Genetic engineering of a separate subunit with a ProteinA tag allows for antibody affinity-based purification using optimized buffer conditions to retain exocyst complex stability. Subsequent proteolytic cleavage of the linker region allows for highly concentrated, purified exocyst complex and has been verified by SDS-PAGE with coomasie blue and silver staining. Negative stain transmission electron microscopy was conducted on the FEI Tecnai G2 TF20 TEM at 200 KeV with images collected at 62,000x nominal magnification. From two-dimensional class averaging of exocyst particles, shaped rod-like structures with dimensions of 10 nm x 28 nm were observed and a calculated conical volume of 2200 nm3 determined. Based on known crystal structure data of Exo70 with a 150 nm3 volume, and assuming other subunits have comparable widths with longer lengths (since Exo70 is the smallest subunit), estimated volume of the entire exocyst complex is approximately 1900 nm3. While efforts are still being made towards localization of the GFP tag and structural characterization by cryo microscopy, we conclude these particles contain the intact exocyst complex with all eight subunits, consistent with biochemical data. |
