Figure 4.

Early endosome fusion is stimulated by GTPγS but is still sensitive to inhibition by wortmannin. (a) Postnuclear supernatants containing avidin- or biotin-HRP–loaded early endosomes were separately preincubated for 10 min at 37°C without (A and B) or with (C and D) 40 μM GTPγS. Correspondingly treated avidin- or biotin-HRP–containing fractions were then combined in a standard fusion assay supplemented with buffer (A and C) or 100 nM wortmannin (B and D) and incubated for 30 min at 37°C. (b) Postnuclear supernatants containing avidin- or biotin-HRP–loaded early endosomes were separately preincubated for various periods of time (Δt) at 37°C with 40 μM GTPγS. Correspondingly treated avidin- or biotin-HRP–containing fractions were then combined in a standard fusion assay in the absence (•) or presence (□) of 100 nM wortmannin and incubated for 30 min at 37°C. The percentage inhibition of the fusion assay signal for each condition is also plotted (▵, dashed line).