The contractile vacuole secretory pathway in Dictyostelium discoideum

grantor: University of TorontoThe endogenous lectin discoidin-I and the Ca2+-dependent cell adhesion molecule DdCAD-1 are soluble cytoplasmic proteins that are synthesized in great abundance during the early phase of 'Dictyostelium discoideum ' development. However, both proteins have been found to associate with the ecto-surface of the plasma membrane. Previous work from our lab has implicated the involvement of contractile vacuoles in transporting DdCAD-1 to the cell surface. It is reported here that discoidin-I is also targeted for secretion via the contractile vacuole. Similar to DdCAD-1, discoidin-I has been found to co-localize with contractile vacuoles by immunofluorescence labelling and subcellular fractionation. Discoidin-I associated with contractile vacuoles is resistant to protease digestion in the absence of detergent, suggesting that the protein is protected within the lumen of these vacuoles. An ' in vitro' reconstitution of protein import into isolated contractile vacuoles was used to demonstrate import of biotinylated discoidin-I into contractile vacuoles and to show that import is time, temperature, and energy dependent. Soluble cytosolic factor(s) were shown to have a positive effect on import and preliminary efforts were made to identify these factors.M.Sc