title: Complementary biochemical approaches applied to the identification of plastidial calmodulin-binding proteins.
abstract: Ca(2+)/Calmodulin (CaM)-dependent signaling pathways play a major role in the modulation of cell responses in eukaryotes. In the chloroplast, few proteins such as the NAD(+) kinase 2 have been previously shown to interact with CaM, but a general picture of the role of Ca(2+)/CaM signaling in this organelle is still lacking. Using CaM-affinity chromatography and mass spectrometry, we identified 210 candidate CaM-binding proteins from different Arabidopsis and spinach chloroplast sub-fractions. A subset of these proteins was validated by an optimized in vitro CaM-binding assay. In addition, we designed two fluorescence anisotropy assays to quantitatively characterize the binding parameters and applied those assays to NAD(+) kinase 2 and selected candidate proteins. On the basis of our results, there might be many more plastidial CaM-binding proteins than previously estimated. In addition, we showed that an array of complementary biochemical techniques is necessary in order to characterize the mode of interaction of candidate proteins with CaM.
authors.affiliation: Université Joseph Fourier Grenoble 1, F-38054 Grenoble, France.
authors.forename: Elisa
authors.initials: E
authors.lastname: Dell'Aglio
authors.forename: Cécile
authors.initials: C
authors.lastname: Giustini
authors.forename: Daniel
authors.initials: D
authors.lastname: Salvi
authors.forename: Sabine
authors.initials: S
authors.lastname: Brugière
authors.forename: Faustine
authors.initials: F
authors.lastname: Delpierre
authors.forename: Lucas
authors.initials: L
authors.lastname: Moyet
authors.forename: Mathieu
authors.initials: M
authors.lastname: Baudet
authors.forename: Daphné
authors.initials: D
authors.lastname: Seigneurin-Berny
authors.forename: Michel
authors.initials: M
authors.lastname: Matringe
authors.forename: Myriam
authors.initials: M
authors.lastname: Ferro
authors.forename: Norbert
authors.initials: N
authors.lastname: Rolland
authors.forename: Gilles
authors.initials: G
authors.lastname: Curien
mesh_terms: D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism*; D020869:Gene Expression Profiling; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis / Q000737:chemistry; D018515:Plant Leaves; D010940:Plant Proteins / Q000378:metabolism; D011485:Protein Binding; D020543:Proteome / Q000032:analysis*; D015398:Signal Transduction; D018724:Spinacia oleracea / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010940:Plant Proteins; D020543:Proteome; D017853:Phosphotransferases (Alcohol Group Acceptor); C021844:NAD kinase; D002118:Calcium