Coronin and vacuolin identify consecutive stages of a late, actin-coated endocytic compartment in Dictyostelium

Cells of the unicellular eukaryote Dictyostelium discoideum take up all their nutrients by endocytosis, Both particle- and fluid-containing vacuoles are transiently surrounded by a cytoskeletal coat [1,2]. When this coat has dissociated, acidification and digestion of the vesicle contents occur, followed by exocytosis of the indigestible remnants after 60-90 minutes, At least nine compartments are needed for mathematical modelling of endocytic transit [3], suggesting that markers associate for only a few minutes with a specific endocytic compartment Among the proteins that have been identified as components of endocytic vesicles are actin, subunits of the V-H(+)ATPase and small GTP-binding proteins of the nab family [4-7]. Using a monoclonal antibody produced against Dictyostelium endocytic vesicles, we have isolated a cDNA corresponding to a novel protein that we have named vacuolin. In order to determine the precise step along the endocytic pathway that involves vacuolin, we generated a fusion protein of the green fluorescent protein (GFP) and vacuolin, GFP-vacuolin-decorated vesicles were identified as a post-lysosomal compartment that acquires endocytic markers shortly before exocytosis. At earlier stages, this post-lysosomal compartment was identified by the binding of a tagged cytoskeletal protein, coronin-GFP. Vacuoles were coated with filamentous actin along the entire post-lysosomal pathway, and the integrity of the actin coat was required for exocytosis.