The Cell Adhesion Molecule DdCAD-1 in <i>Dictyostelium</i> Is Targeted to the Cell Surface by a Nonclassical Transport Pathway Involving Contractile Vacuoles

<jats:p>DdCAD-1 is a 24-kD Ca2+-dependent cell– cell adhesion molecule that is expressed soon after the initiation of development in Dictyostelium cells. 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 hydrophobic signal peptide or any predicted transmembrane domain, suggesting that it may be presented on the cell surface via a nonclassical transport mechanism. Here we report that DdCAD-1 is transported to the cell surface via contractile vacuoles, which are normally involved in osmoregulation. Immunofluorescence microscopy and subcellular fractionation revealed a preferential association of DdCAD-1 with contractile vacuoles. Proteolytic treatment of isolated contractile vacuoles degraded vacuole-associated calmodulin but not DdCAD-1, demonstrating that DdCAD-1 was present in the lumen. The use of hyperosmotic conditions that suppress contractile vacuole activity led to a dramatic decrease in DdCAD-1 accumulation on the cell surface and the absence of cell cohesiveness. Shifting cells back to a hypotonic condition after hypertonic treatments induced a rapid increase in DdCAD-1–positive contractile vacuoles, followed by the accumulation of DdCAD-1 on the cell membrane. 7-chloro-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. Furthermore, an in vitro reconstitution system was established, and isolated contractile vacuoles were shown to import soluble DdCAD-1 into their lumen in an ATP-stimulated manner. Taken together, these data provide the first evidence for a nonclassical protein transport mechanism that uses contractile vacuoles to target a soluble cytosolic protein to the cell surface.</jats:p>