Figure 1.

Wortmannin inhibition of XTPγS-REP-1^d136n-dependent early endosome fusion. (a) Postnuclear supernatants containing avidin- or biotin-HRP–loaded early endosomes were aliquoted and separately preincubated at 37°C for 10 min with REP-1 complex buffer (A and B); 100 nM REP-1/rab5^D136N (C and D); 100 nM REP-1/rab5^D136N, 0.5 mM XTPγS (E and F). Correspondingly treated avidin- or biotin-HRP–containing fractions were then combined in a standard fusion assay and incubated at 37°C for 30 min in the presence (B, D, and F) or absence (A, C, and E) of 100 nM wortmannin. (b) Postnuclear supernatants containing avidin- or biotin-HRP–loaded early endosomes were aliquoted and separately preincubated for 10 min at 37°C with REP-1 complex buffer (□) or 100 nM REP-1/rab5^D136N,0.5 mM XTPγS(•). Correspondingly treated avidin- or biotin-HRP–containing fractions were then combined in a standard fusion assay and incubated at 37°C for 30 min with the indicated concentrations of wortmannin.