Figure 4. TRAPPI and Sar1p-GTP cannot bind to Sec23p simultaneously.

a, GST-Sec23p beads were incubated with 10 nM of His₆-Δ23Sar1p-GTPγS and increasing concentrations of TRAPPI (top), or 10 nM TRAPPI and increasing concentrations of His₆-Δ23Sar1p-GTPγS (bottom). b, mBet3 is on COPII vesicles formed with GTP (−Sar1), but not GMP-PNP (+Sar1). Vesicles formed in vitro with GTP or GMP-PNP were immunoisolated with an antibody against the cargo VSV-G-myc and blotted for mBet3 and mSec23. The data was normalized to cargo yield (see METHODS). N=2, bars show the range. c, In vitro transport was performed with increasing concentrations of IC261 and fusion was measured. Error bars represent S.D., N=4. d, The in vitro transport assay was performed as in Fig. 4c with IC261 and budding was measured. Error bars represent S.D., N=3 (*P<0.05, **P<0.01,***P<0.001 Student’s t test)