Journal
PLANT PHYSIOLOGY AND BIOCHEMISTRY
Volume 49, Issue 1, Pages 18-32Publisher
ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER
DOI: 10.1016/j.plaphy.2010.09.015
Keywords
Fruit ripening; Membrane; Senescence; Calcium; Phosphatidylinositol phosphate; Fluorescence resonance energy transfer; Lipid; Immunohistochemistry
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Funding
- Natural Sciences and Engineering Research Council of Canada
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C2 domains exist as highly conserved N-terminal or C-terminal calcium- and lipid-binding motifs comprising nearly 130 amino acids, responsible for recruiting proteins to the membrane during signal transduction. In this study, the sequence corresponding to the N-terminal 164 amino acids of a full length cDNA of phospholipase D alpha from tomato fruit was cloned in pET28(b) vector and expressed in E. coli as a His-tagged protein. Recombinant C2 domain showed micromolar affinity towards Ca(++) with a maximum of 2 high affinity binding sites. Interaction of C2 domain with synthetic unilamellar vesicles, evaluated by protein- lipid fluorescence resonance energy transfer, showed maximum affinity towards phosphatidic acid, and virtually no binding with phosphatidylcholine. The binding towards phosphoinositides was reduced with increasing degree of phosphorylation. Acid- and chaotropic salt- titrations indicated an electrostatic, rather than a hydrophobic mode of interaction between C2 domain and the phospholipid vesicles. Conformational analyses of the recombinant C2 domain showed a much longer calcium binding loop region, a far less electropositive phosphoinositide-binding region, unique calcium binding pockets with high electro-negativity, and other features that are distinct from the typical C2 domains of phospholipase A2 and Protein kinase C alpha, signifying the uniqueness of Phospholipase D alpha in fruit developmental events. (C) 2010 Elsevier Masson SAS. All rights reserved.
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