Dynamics of the COPII coat with GTP and stable analogues

We have developed an assay to monitor the assembly of the COPII coat onto l iposomes in real time. We show that with Sar1pGTP bound to liposomes, a sin gle round of assembly and disassembly of the COPII coat lasts a few seconds . The two large COPII complexes Sec23/24p and Sec13/31p bind almost instant aneously (in less than 1 s) to Sar1pGTP-doped liposomes. This binding is fo llowed by a fast (less than 10 s) disassembly due to a 10-fold acceleration of the GTPase-activating protein activity of Sec23/24p by the Sec13/31p co mplex. Experiments with the phosphate analogue BeFx suggest that Sec23/24p provides residues directly involved in GTP hydrolysis on Sar1p.