Cell Adhesion Molecule DdCAD-1 Is Imported into Contractile Vacuoles by Membrane Invagination in a Ca2+- and Conformation-dependent Manner

Abstract

The cadA gene in Dictyostelium encodes a Ca 2؉ -dependent cell adhesion molecule DdCAD-1 that contains two ␤-sandwich domains. DdCAD-1 is synthesized in the cytoplasm as a soluble protein and then transported by contractile vacuoles to the plasma membrane for surface presentation or secretion. DdCAD-1-green fluorescent protein (GFP) fusion protein was expressed in cadA-null cells for further investigation of this unconventional protein transport pathway. Both morphological and biochemical characterizations showed that DdCAD-1-GFP was imported into contractile vacuoles. Time-lapse microscopy of transfectants revealed the transient appearance of DdCAD-1-GFP-filled vesicular structures in the lumen of contractile vacuoles, suggesting that DdCAD-1 could be imported by invagination of contractile vacuole membrane. To assess the structural requirements in this transport process, the N-terminal and C-terminal domains of DdCAD-1 were expressed separately in cells as GFP fusion proteins. Both fusion proteins failed to enter the contractile vacuole, suggesting that the integrity of DdCAD-1 is required for import. Such a requirement was also observed in in vitro reconstitution assays using His 6 -tagged fusion proteins and purified contractile vacuoles. Import of DdCAD-1 was compromised when two of its three Ca 2؉ -binding sites were mutated, indicating a role for Ca 2؉ in the import process. Spectral analysis showed that mutations in the Ca 2؉ -binding sites resulted in subtle conformational changes. Indeed, proteins with altered conformation failed to enter the contractile vacuole, suggesting that the import signal is somehow integrated in the three-dimensional structure of DdCAD-1.

ATP-Binding Cassette Transporter B4 Anchors the Cell Adhesion Molecule DdCAD-1 to Cell Membrane in Dictyostelium discoideum

“…His 6 -tagged DdCAD-1 was bound onto the Ni-NAT beads through the chelating interaction between histidine residues and the nickel ions immobilized on beads. Elution buffer containing 250 mM imidazole was used to elute the bound His 6 -DdCAD-1 from beads [7].…”

“…5b). An alternative route for DdCAD-1 import into contractile vacuoles is through the invagination of the vacuolar membrane [7].…”

“…DdCAD-1 contains two domains, N-terminal domain has homophilic binding function, while C-terminal domain is responsible for tethering DdCAD-1 on cell membrane [4,5]. DdCAD-1 is transported to cell surface by contractile vacuoles [6], and time-lapse images show that some of DdCAD-1 is imported to contractile vacuoles by membrane invagination [7]. Moreover, DdCAD-1 can form capping structure on the cell surface by antibodies crosslinking experiment, which also demonstrates that DdCAD-1 is anchored on cell surface by certain membrane receptor(s) [8].…”

The cell adhesion molecule DdCAD-1 regulates morphogenesis through differential spatiotemporal expression inDictyostelium discoideum

“…S1 in the supplementary material). Mutations in cadA were generated as described previously (Sriskanthadevan et al, 2009) and all constructs were sequenced from both ends. Plasmid DNAs were electroporated into cadA -cells (Pang et al, 1999).…”

“…DdCAD-1 is unique among CAMs because it is synthesized as a soluble protein and transported to the plasma membrane by contractile vacuoles (Sesaki et al, 1997;Sriskanthadevan et al, 2009). DdCAD-1 contains two distinct domains with -sandwich architecture.…”