MEMBRANE-POTENTIAL MEDIATES H-ATPASE DEPENDENCE OF DEGRADATIVE PATHWAY ENDOSOMAL FUSION()

In some epithelial cell lines, the uptake and degradation of proteins is so pronounced as to be regarded as a specialized function known as ''degradative endocytosis.'' The endosomal pathways of the renal proxi mal tubule and the visceral yolk sac share highly specialized structur es for ''degradative endocytosis.'' These endosomal pathways also have a unique distribution of their H+-ATPase, predominantly in the subapi cal endosomal pathway. Previous studies provide only indirect evidence that H+-ATPases participate in endosomal fusion events: formation of vesicular intermediates between early and late endosomes is H+-ATPase dependent in baby hamster kidney cells, and H+-ATPase subunits bind fu sion complex proteins in detergent extracts of fresh rat brain. To det ermine directly whether homotypic endosomal fusion is H+-ATPase depend ent, we inhibited v-type H+-ATPase during flow cytometry and cuvette-b ased fusion assays reconstituting endosomal fusion in vitro. We report that homotypic fusion in subapical endosomes derived from rat renal c ortex, and immortalized visceral yolk sac cells in culture, is inhibit ed by the v-type H+-ATPase specific inhibitor bafilomycin Al. Inhibiti on of fusion by H+-ATPase is mediated by the membrane potential as col lapsing the pH gradient with nigericin had no effect on homotypic endo somal fusion, while collapsing the membrane potential with valinomycin inhibited endosomal fusion. Utilizing an in vitro reconstitution assa y this data provides the first direct evidence for a role of v-type Hc -ATPase in mammalian homotypic endosomal fusion.