Functional Characterization of the N-Terminal C2 Domain from Arabidopsis thaliana Phospholipase Dα and DβReport as inadecuate

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BioMed Research International - Volume 2016 2016, Article ID 2721719, 15 pages -

Research ArticleInstitut de Chimie et de Biochimie Moléculaires et Supramoléculaires ICBMS, Univ Lyon, Université Lyon 1, UMR 5246 CNRS, Métabolisme, Enzymes et Mécanismes Moléculaires MEM

, F-69622 Villeurbanne cedex, France

Received 13 July 2016; Revised 6 October 2016; Accepted 27 October 2016

Academic Editor: Pengjun Shi

Copyright © 2016 Renaud Rahier et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Most of plant phospholipases D PLD exhibit a C2-lipid binding domain of around 130 amino acid residues at their N-terminal region, involved in their Ca

-dependent membrane binding. In this study, we expressed and partially purified catalytically active PLDα from Arabidopsis thaliana AtPLDα in the yeast Pichia pastoris. The N-terminal amino acid sequence of the recombinant AtPLDα was found to be NVEETIGV and thus to lack the first 35 amino acid belonging to the C2 domain, as found in other recombinant or plant purified PLDs. To investigate the impact of such a cleavage on the functionality of C2 domains, we expressed, in E. coli, purified, and refolded the mature-like form of the C2 domain of the AtPLDα along with its equivalent C2 domain of the AtPLDβ, for the sake of comparison. Using Förster Resonance Energy Transfer and dot-blot assays, both C2 domains were shown to bind phosphatidylglycerol in a Ca

-independent manner while phosphatidic acid and phosphatidylserine binding were found to be enhanced in the presence of Ca

. Amino acid sequence alignment and molecular modeling of both C2 domains with known C2 domain structures revealed the presence of a novel Ca

-binding site within the C2 domain of AtPLDα.

Author: Renaud Rahier, Alexandre Noiriel, and Abdelkarim Abousalham



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