FIGURE 5:

PI(4)P is required at the Golgi for fusion with COPII vesicles. Microsomes containing [³⁵S]gpαf were treated with 50 μM Sac1^WT or buffer (mock) at 12°C for 30 min. Treated microsomes were then used to generate COPII vesicles, and vesicles were purified by flotation on Nycodenz density gradients. Isolated vesicles were then incubated in two-stage fusion reactions with untreated semi-intact cell acceptor membranes as in Figure 1B.