TY - JOUR
T1 - Purification and functional properties of yeast Sec12 GEF
AU - Futai, Eugene
AU - Schekman, Randy
N1 - Funding Information:
We thank Crystal Chan and Robert Lesch for COPII proteins and Matthew Welsh and David G. Drubin for sharing their equipment. We thank Chris Fromme for improving the manuscript and Bruno Antonny for advice on kinetic analysis of real‐time assays. We thank members of the Schekman lab for discussion and encouragement. This work was supported by the HHMI (R.S.) and postdoctoral research fellowships from JSPS and ACS (E.F.).
PY - 2005
Y1 - 2005
N2 - 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.
AB - 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.
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M3 - Review article
C2 - 16413259
AN - SCOPUS:30544453498
SN - 0076-6879
VL - 404
SP - 74
EP - 82
JO - Methods in Enzymology
JF - Methods in Enzymology
M1 - 8
ER -