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 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.
Sesaki
H
H
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Wong
E F
EF
Siu
C H
CH
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002135:Calcium-Binding Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004023:Dictyostelium / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006982:Hypertonic Solutions; D007038:Hypotonic Solutions; D014617:Vacuoles / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D015815:Cell Adhesion Molecules; C100604:DdCAD-1 protein, Dictyostelium discoideum; D006982:Hypertonic Solutions; D007038:Hypotonic Solutions; D009327:4-Chloro-7-nitrobenzofurazan