Fig. 2. Effect of cytosol and ATP on PCTV and protein vesicle budding from ER. (A) ER containing ³H-TAG was incubated in the presence (+) or absence (-) of intestinal cytosol. A continuous sucrose gradient (0.1-1.15 M) was used to isolate the PCTVs. The total ³H-dpm from each fraction (150 µl of 500 µl total volume) was counted. (B) A similar experiment to A was performed with (+) or without (-) ATP and an ATP generating system. The gradient was resolved as in A and total ³H-dpm determined. In A and B only fractions 1-20 of 70 total fractions (total tube volume, 35 ml) are shown since no ³H-dpm above baseline were found in fractions 21-70. (C) ER containing ¹⁴C-TAG and ³H-protein were incubated with and without cytosol in the presence of an ATP-generating system. The reaction mix was resolved on the sucrose gradient using 11 ml centrifuge tubes and fractions of 0.5 ml collected.