In order to reconstitute the generation of COPII vesicles from synthetic liposomes, the minimum requirements are the coat components, Sar1p GTPase, Sec23/24p, Sec13/31p, and a nonhydrolyzable GTP analog such as GMP-PNP. However, in the presence of GTP, nucleotide hydrolysis by Sar1p renders the coat insufficiently stable to sustain vesicle budding. Sar1p GTPase activity was activated by the Sec23/24p GTPase-activating protein (GAP), and further accelerated 10-fold by Sec13/31p. In order to study GTP-dependent budding, we introduced the Sar1p guanine nucleotide exchange factor (GEF), Sec12p. We evaluated Sar1p activation by Sec12p and the dynamics of coat assembly and disassembly in the presence of both Sec12p and Sec23/24p. The cytoplasmic domain of Sec12p activated Sar1p with a turnover 10-fold higher than the GAP activity of Sec23p in the presence of Sec13/31p. As a result, the entire COPII coat remains stable in the presence of GTP. Here, we describe methods to purify Sec12p, real-time fluorescence assays to evaluate COPII coat formation, and the relevant kinetic analyses.
|Number of pages||9|
|Journal||Methods in enzymology|
|Publication status||Published - 2005|
ASJC Scopus subject areas
- Molecular Biology