Cell adhesion molecule DdCAD-1 is imported into contractile vacuoles by membrane invagination in a Ca2+- and conformation-dependent manner.
The cadA gene in Dictyostelium encodes a Ca(2+)-dependent cell adhesion molecule DdCAD-1 that contains two beta-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.
Sriskanthadevan
Shrivani
S
Banting and Best Department of Medical Research and Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Lee
Teresa
T
Banting and Best Department of Medical Research and Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Lin
Zhi
Z
Department of Biological Sciences, National University of Singapore, Singapore 117543.
Yang
Daiwen
D
Department of Biological Sciences, National University of Singapore, Singapore 117543.
Siu
Chi-Hung
CH
Banting and Best Department of Medical Research and Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Electronic address: chi.hung.siu@utoronto.ca.
D002118:Calcium / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000235:genetics / Q000378:metabolism*
D015815:Cell Adhesion Molecules; D011993:Recombinant Fusion Proteins; D002118:Calcium