Fig. 4. Transport to the Golgi and delivery to the Golgi lumen of newly synthesized ¹⁴C-TAG and ³H-apoB-48 in PCTVs. (A) PCTVs (150 µg prot) containing ¹⁴C-TAG and ³H-apoB-48 were incubated with non-radiolabeled Golgi (300 µg prot) in the presence (+) or absence (-) of cytosol (0.5 mg prot). The Golgi was separated from PCTVs and the Golgi ¹⁴C-TAG extracted. The Golgi ³H-apoB-48 was isolated by immunoprecipitation. Of the total ¹⁴C-TAG-dpm and ³H-apoB-48-dpm, 28% and 25%, respectively, were delivered to the Golgi during the incubation. (B) After the fusion reaction, the Golgi was separated from PCTV on a sucrose gradient and incubated with or without carbonate (100 mM, pH 11) as indicated to release the chylomicrons. Chylomicron ¹⁴C-TAG was extracted, and its ³H-apoB-48 isolated by immunoprecipitation. The data are the means of three experiments. (C) ³H-dpm-PCTVs (150 µg prot, 30,640 dpm) were incubated with Golgi membranes (300 µg prot). The Golgi were isolated and treated with (+) or without (-) trypsin (0.5 mg/ml final concentration) for 1 hour at 4°C. Trypsin inhibitors were added and the Golgi isolated. ³H-dpm was determined for each fraction. (Inset) 30 µg protein from fractions 18-21 from the incubations with or without trypsin were loaded onto SDS-PAGE and immunoblotted for GOS28. (D) The fusion of ³H-TAG-PCTV generated in native cytosol (Normal PCTVs) or Sar1-depleted cytosol (COPII dep PCTV) with Golgi. The Golgi was isolated and ³H-TAG extracted. The data are the mean±1 s.e.m. (n=3). For details, see Materials and Methods.