SUMMARY
The exocyst is an octameric protein complex that is conserved throughout the Eukaryotes and is essential for life. It tethers exocytic vesicles to the plasma membrane prior to fusion. Exocyst assembly and delivery mechanisms remain unclear, especially in mammalian cells. To address these issues we tagged endogenous exocyst subunits with sfGFP or Halo using Cas9 gene editing, to create single and double knock-in lines of NMuMG mammary epithelial cells and used high speed imaging to capture the dynamics of exocyst assembly and disassembly. Surprisingly, we find that as in yeast the mammalian exocyst is comprised of 2 subcomplexes, but these can each associate independently with the plasma membrane and are in a dynamic equilibrium. Moreover, although all subunits arrive at a vesicle fusion site at similar times (~14 sec prior to fusion), departure of one subunit, Sec3, occurs ~1 sec prior to fusion, whereas all others depart just after fusion. Single molecule counting indicates ~9 exocyst complexes associated with each vesicle. These data reveal the mammalian exocyst to be an unexpectedly dynamic two-part complex, and we propose a new model for its assembly and disassembly.
