Figure 6.

Increasing membrane concentrations of his₆ rab5 with or without GTPγS partially protect early endosome fusion from wortmannin inhibition. (a) Postnuclear supernatants containing avidin- or biotin-HRP—loaded early endosomes were separately preincubated for 10 min at 37°C with 60 μM GTPγS and 0, 50, 200, 500, or 1000 nM REP-1/rab5. Correspondingly treated avidin or biotin-HRP—containing fractions were then combined in a standard fusion assay for 40 min at 37°C in the presence (□) or absence (•) of wortmannin (100 nM). (b) Postnuclear supernatants were incubated as in Figure 6a. The samples were centrifuged and 13 μg of the pelleted fractions were loaded on a 13% SDS-PAGE gel. Rab5 was detected by Western blotting using a rab5 monoclonal antibody. The position of BHK-21 rab5 and his₆-tagged native rab5 is shown. (c) Postnuclear supernatants containing avidin- or biotin-HRP—loaded early endosomes were separately preincubated without added nucleotide for 10 min at 37°C with 0,50, 200, 500, or 1000 nM REP-1/rab.5 Correspondingly treated avidin or biotin-HRP containing fractions were then combined in a starndard fusion assay for 40 min at 37°C in the presence (□) or absence (•) of wortmannin (100 nM).