Journal
FEBS JOURNAL
Volume 280, Issue 8, Pages 1795-1806Publisher
WILEY-BLACKWELL
DOI: 10.1111/febs.12203
Keywords
calmodulin binding protein; cell adhesion molecule; contractile vacuoles; Dictyosteliumdiscoideum; unconventional protein transport
Categories
Funding
- Canadian Institutes of Health Research [FRN-6140]
- Ontario Graduate Scholarship
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The Ca2+-dependent cellcell adhesion molecule DdCAD-1, encoded by the cadA gene of Dictyosteliumdiscoideum, 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 Ca2+-dependent manner, and co-localizes with DdCAD-1 on the surface of contractile vacuoles. Bioinformatics analysis revealed multiple calmodulin-binding motifs in DdCAD-1. Co-immunoprecipitation and pull-down studies showed that only Ca2+-bound calmodulin was able to bind DdCAD-1. Structural integrity of DdCAD-1, but not the native conformation, was required for its interaction with calmodulin. To investigate the role of calmodulin in the import of DdCAD-1 into contractile vacuoles, an invitro 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 DdCAD-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 DdCAD-1. Together, the data show that calmodulin forms a complex with DdCAD-1 and promotes the docking and import of DdCAD-1 into contractile vacuoles. Structured digital abstract CaM 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)
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