Protein secretion is executed by a cellular pathway involving the delivery of membrane and soluble secretory proteins in vesicles that capture newly-synthesized proteins assembled in the endoplasmic reticulum (ER) and sorted in the Golgi apparatus. Vesicles fuse with the plasma membrane resulting in the discharge of soluble molecules to the cell exterior and integration of vesicle membrane proteins and lipids in the cell surface. Baker’s yeast cells grow by vesicle fusion and secretion at the tip of the daughter bud. A genetic dissection of this process was performed with temperature sensitive conditional mutants blocked at one of several stations in the secretory pathway.
Secretion mutants that block protein exit from the endoplasmic reticulum define genes involved in the formation, targeting and fusion of a small vesicle intermediate. SEC genes corresponding to the mutants defective in vesicle budding define the cytoplasmic machinery responsible for transport vesicle morphogenesis. A biochemical reaction that reproduces ER vesicle budding was reconstituted with gently-broken yeast cells and pure recombinant Sec proteins required in vivo for this budding event. The Sec proteins assemble on the ER membrane in the presence of GTP which activates a small GTPase, Sar1, initiating the formation of a coat protein complex called COPII.
In this talk, Randy Schekman describes a genetic screen in yeast and then follow-up studies in vitro (in the test tube).
- Why would a scientist reconstruct vesicle budding in vitro when this phenomenon can be readily observed in live cells?
- What are some of the advantages of using biochemical techniques to study the secretory pathway?