Premium
C a 2+ ‐calmodulin interacts with D d CAD ‐1 and promotes D d CAD ‐1 transport by contractile vacuoles in D ictyostelium cells
Author(s) -
Sriskanthadevan Shrivani,
Brar Simuran K.,
Manoharan Kumararaaj,
Siu ChiHung
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12203
Subject(s) - calmodulin , vacuole , immunoprecipitation , calmodulin binding proteins , microbiology and biotechnology , dictyostelium discoideum , egta , biology , biochemistry , chemistry , calcium , gene , enzyme , organic chemistry , cytoplasm
The C a 2+ ‐dependent cell–cell adhesion molecule Dd CAD ‐1, encoded by the cadA gene of D ictyostelium discoideum , is synthesized at the onset of development as a soluble protein and then transported to the plasma membrane by contractile vacuoles. Calmodulin associates with contractile vacuoles in a Ca 2+ ‐dependent manner, and co‐localizes with Dd CAD ‐1 on the surface of contractile vacuoles. Bioinformatics analysis revealed multiple calmodulin‐binding motifs in Dd CAD ‐1. Co‐immunoprecipitation and pull‐down studies showed that only C a 2+ ‐bound calmodulin was able to bind Dd CAD ‐1. Structural integrity of D d CAD ‐1, but not the native conformation, was required for its interaction with calmodulin. To investigate the role of calmodulin in the import of Dd CAD ‐1 into contractile vacuoles, an in vitro import assay consisting of contractile vacuoles derived from cadA − cells and recombinant proteins was employed. Prior stripping of the bound calmodulin from contractile vacuoles by EGTA impaired import of Dd CAD ‐1, which was restored by addition of exogenous calmodulin. The calmodulin antagonists W‐7 and compound 48/80 blocked the binding of calmodulin onto stripped contractile vacuoles, and inhibited the import of Dd CAD ‐1. Together, the data show that calmodulin forms a complex with Dd CAD ‐1 and promotes the docking and import of Dd CAD ‐1 into contractile vacuoles. Structured digital abstractCaM physically interacts with DdCAD-1 by pull down (View Interaction: 1 , 2 ) DdCAD-1 binds to CaM by far western blotting ( View interaction ) DdCAD-1 physically interacts with CaM by anti bait coimmunoprecipitation ( View interaction )