In vitro, vesicle strings developed to remarkable dimensions of dozens of connected vesicles, several micrometers in total length (Fig. 2b–d), which supports the notion of Bannykh et al.15 that vesicle separation fails in the absence of GTP hydrolysis. To exclude extraneous effects of non-hydrolysable GTP analogs, we used hydrolysable GTP and replaced wtSar1p by Sar1p(H77L) (Fig. 2j). The intrinsic GTPase activity of this mutant is similarly low as that of wtSar1p, but stimulation of the GTPase activity by the Sec23/24p complex is reduced from about 9-fold to 2-fold3. The lowered GTPase activity of the mutant in the presence of the full COPII complex proved sufficient for the formation of rigid, multibudded extensions (Fig. 2j, Movie S3, Fig. 3k), as seen with the non-hydrolysable GTP analog.