Characterization of trypsin-sensitive factor(s) required for endosome-endosome fusion

Fusion of endosomes appears to be required at early steps of receptor-mediated endocytosis. These fusion events have been reconstituted using a cell-free assay and have been shown to require both cytosolic and membrane-associated proteins. We report here that trypsinization of endosomes completely inhibited fusion. Addition of untreated cytosol cannot restore fusion of trypsinized endosomes. However, fusion activity is restored by the addition of either untreated vesicles or a high salt extract containing peripheral membrane proteins (KE). KE contains both the membrane-associated factor(s) required for the reconstitution of fusion using trypsinized endosomes and the factors that are normally provided by the cytosol. The restorative activity of KE was sensitive to trypsin treatment or incubation at 100 degrees C, but was largely N-ethylmaleimide (NEM)-resistant. This and other criteria demonstrated that the trypsin-sensitive factor is distinct from N-ethylmaleimide-sensitive factor (NSF), an NEM-sensitive protein involved in vesicular fusion, and from other known factors that may participate in membrane fusion events. Preliminary fractionation studies indicate that the restorative activity of KE is associated with one or more high molecular weight proteins. The present study indicates that a novel trypsin-sensitive protein(s) is involved in endosome-endosome fusion. This factor is membrane-associated and is not found in an active form in cytosol as prepared.