Vesicle release is regulated by endogenous Sar1 GTPase activity and requires functional NH2-terminal amphipathic domain. (A) Inhibition of endogenous Sar1 GTPase activity inhibits COPII vesicle release. VSV-G–containing membranes were incubated in the presence of cytosol for 30 min on ice (lane 1) or at 32°C (lanes 2–7) in the absence (lanes 1 and 2) or presence of increasing concentrations of GTP-γ-S as indicated. At the end of incubation, the vesicle fraction (H) was separated from the donor membranes (M) by differential centrifugation without trituration and analyzed by Western blotting as indicated. (B) COPII components are not limiting under conditions inhibitory to vesicle release. The vesicle formation reaction was carried in the absence or presence of GTP-γ-S (100 μM). At the end of incubation the vesicle fraction was separated from the membrane fraction. The supernatant of the vesicle fraction was collected and analyzed for available COPII component Sec23. Quantitation of the amount of VSV-G in the vesicle fraction (left) and COPII Sec23 subunit remaining in the supernatant of the vesicle fraction (right) averaged from three independent experiments ± SEM. (C) Cargo-free COPII vesicles are not produced when Sar1 GTPase activity is inhibited. Vesicles generated as described in A, in the presence or absence of 100 μM GTP-γ-S, were separated from donor membranes by centrifugation and floated into sucrose gradients. Fractions 1–4 (collected from the top of the gradient) contain floated vesicles (VSV-G– and Bet1-containing fractions). The presence of VSV-G, Sec23, and Bet1 in the vesicle fraction was determined as indicated. (D) Sar1FPF (Y9F, G11P, S14F) does not support efficient vesicle release. In the upper panel, VSV-G–containing membranes were incubated (40 μl final volume) with limiting cytosol in the presence of Sar1 wt or Sar1FPF (2.5 μg each) on ice or at 32°C for 30 min as indicated. At the end of incubation the vesicle fraction was prepared without physical trituration and analyzed by Western blotting. For the trituration assay (lower panel), membranes were incubated (40 μl final volume) with limiting cytosol in the presence of Sar1 wt or Sar1FPF (2 μg each) for 15 min on ice or at 32°C as indicated. The vesicle fraction was prepared with physical trituration and analyzed by Western blotting.
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