THE CELL-ADHESION MOLECULE DDCAD-1 IN DICTYOSTELIUM IS TARGETED TO THE CELL-SURFACE BY A NONCLASSICAL TRANSPORT PATHWAY INVOLVING CONTRACTILE VACUOLES

DdCAD-1 is a 24-kD Ca2+-dependent cell-cell adhesion molecule that is expressed soon after the initiation of development in Dictyostelium ce lls. DdCAD-1 is present on the cell surface as well as in the cytosol. However, the deduced amino acid sequence of DdCAD-1 lacks a hydrophob ic signal peptide or any predicted transmembrane domain, suggesting th at it may be presented on the cell surface via a nonclassical transpor t mechanism. Here we report that DdCAD-1, is transported to the cell s urface via contractile vacuoles, which are normally involved in osmore gulation. Immunofluorescence microscopy and subcellular fractionation revealed a preferential association of DdCAD-1 with contractile vacuol es. Proteolytic treatment of isolated contractile vacuoles degraded va cuole-associated calmodulin but not DdCAD-1, demonstrating that DdCAD- 1 was present in the lumen. The use of hyperosmotic conditions that su ppress contractile vacuole activity led to a dramatic decrease in DdCA D-1 accumulation on the cell surface and the absence of cell cohesiven ess. Shifting cells back to a hypotonic condition after hypertonic tre atments induced a rapid increase in DdCAD-1-positive contractile vacuo les, followed by the accumulation of DdCAD-1 on the cell membrane. 7-c hloro-4-nitrobenzo-2-oxa-1,3-diazole, a specific inhibitor of vacuolar -type H+-ATPase and thus of the activity of contractile vacuoles, also inhibited the accumulation of DdCAD-1 on the cell surface. Furthermor e, an in vitro reconstitution system was established, and isolated con tractile vacuoles were shown to import soluble DdCAD-1 into their lume n in an ATP-stimulated manner. Taken together, these data provide the first evidence for a nonclassical protein transport mechanism that use s contractile vacuoles to target a soluble cytosolic protein to the ce ll surface.