Fig. 8. The effect of Sar1 antibodies and Sar1-depleted cytosol on PCTV (A,B,C, E) and protein vesicle (D,F) budding from ER. (A) The effect of Sar1-depleted cytosol (0.8 mg protein) on PCTV-budding. Native ER was incubated with (+) or without (-) cytosol and compared with ER incubated with Sar1-depleted cytosol (0.8 mg protein). Total ³H-dpm was determined for each fraction. (B) Cytosol (1 mg protein) was (+) or was not (-) included in the budding assay. Pre-immune IgG was added to the native cytosol (+ Cytosol + IgG). Native ER was incubated with cytosol to which Sar1 antibodies had been added (ER + Sar1 Ab.) Native ER was treated with 2 M urea and incubated with cytosol to which Sar1 antibody had been added (Urea ER + Sar1 Ab). The total ³H-dpm is shown on the ordinate. For A and B, 35 ml centrifuge tubes were used. Data from fractions 1-20 are shown since fractions 21 to 70 had no ³H-dpm over baseline. (C,D) The effect of Sar1-depleted cytosol on PCTV and protein vesicle budding. ER containing ¹⁴C-TAG and ³H-protein was immunodepleted of Sar1 (+ Sar1 dep cy), mock depleted cytosol (Moc dep cy) or no cytosol (- Cy). The reaction mix was resolved on a sucrose gradient using 11 ml centrifuge tubes and 0.5 ml fractions obtained. The ¹⁴C-dpm-TAG (C) and TCA precipitable ³H-dpm-protein (D) were determined for each fraction. (E,F) The effect of recombinant Sar1 protein on PCTV and protein vesicle budding in the presence of Sar1-depleted cytosol. Experiments similar to C and D were performed using ¹⁴C-TAG and ³H-protein-labeled ER incubated with no cytosol (- Cy), mock depleted cytosol (+ Cy), Sar1-depleted cytosol (Sar1 dep cy) or Sar1-depleted cytosol supplemented with recombinant Sar1 protein (Sar1 rep cy). ¹⁴C-TAG-dpm (E) and ³H-protein-dpm (F) were determined.