CORONIN AND VACUOLIN IDENTIFY CONSECUTIVE STAGES OF A LATE, ACTIN-COATED ENDOCYTIC COMPARTMENT IN DICTYOSTELIUM

Cells of the unicellular eukaryote Dictyostelium discoideum take up al l 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 a fter 60-90 minutes, At least nine compartments are needed for mathemat ical modelling of endocytic transit [3], suggesting that markers assoc iate for only a few minutes with a specific endocytic compartment Amon g the proteins that have been identified as components of endocytic ve sicles are actin, subunits of the V-H(+)ATPase and small GTP-binding p roteins of the nab family [4-7]. Using a monoclonal antibody produced against Dictyostelium endocytic vesicles, we have isolated a cDNA corr esponding to a novel protein that we have named vacuolin. In order to determine the precise step along the endocytic pathway that involves v acuolin, we generated a fusion protein of the green fluorescent protei n (GFP) and vacuolin, GFP-vacuolin-decorated vesicles were identified as a post-lysosomal compartment that acquires endocytic markers shortl y before exocytosis. At earlier stages, this post-lysosomal compartmen t was identified by the binding of a tagged cytoskeletal protein, coro nin-GFP. Vacuoles were coated with filamentous actin along the entire post-lysosomal pathway, and the integrity of the actin coat was requir ed for exocytosis.