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 Ca2+-dependent cell adhesion molecule DdCAD-1 that contains two β-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 His6-tagged fusion proteins and purified contractile vacuoles. Import of DdCAD-1 was compromised when two of its three Ca2+-binding sites were mutated, indicating a role for Ca2+ in the import process. Spectral analysis showed that mutations in the Ca2+-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. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.