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Phosphorylation and subsequent interaction with 14-3-3 proteins regulate plastid glutamine synthetase in Medicago truncatula

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Abstract

In this report we demonstrate that plastid glutamine synthetase of Medicago truncatula (MtGS2) is regulated by phosphorylation and 14-3-3 interaction. To investigate regulatory aspects of GS2 phosphorylation, we have produced non-phosphorylated GS2 proteins by expressing the plant cDNA in E. coli and performed in vitro phosphorylation assays. The recombinant isoenzyme was phosphorylated by calcium dependent kinase(s) present in leaves, roots and nodules. Using an (His)6-tagged 14-3-3 protein column affinity purification method, we demonstrate that phosphorylated GS2 interacts with 14-3-3 proteins and that this interaction leads to selective proteolysis of the plastid located isoform, resulting in inactivation of the isoenzyme. By site directed mutagenesis we were able to identify a GS2 phosphorylation site (Ser97) crucial for the interaction with 14-3-3s. Phosphorylation of this target residue can be functionally mimicked by replacing Ser97 by Asp, indicating that the introduction of a negative charge contributes to the interaction with 14-3-3 proteins and subsequent specific proteolysis. Furthermore, we document that plant extracts contain protease activity that cleaves the GS2 protein only when it is bound to 14-3-3 proteins following either phosphorylation or mimicking of phosphorylation by Ser97Asp.

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Abbreviations

GS:

glutamine synthetase

GS2:

plastid GS

NR:

nitrate reductase

Ni-NTA:

nickel-nitrilotriacetic acid

6x his-tag:

six histidine tag

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Acknowledgments

We gratefully acknowledge Dr. Carol Mackintosh (MRC unit, University of Dundee, UK) for providing anti-14-3-3 antibodies. We are also extremely grateful to Michel Rossignol and Giselle Borderie (IFR40, Toulouse, France) for expert assistance in 2D electrophoresis. We are also grateful to Jorge Azevedo and Pedro Pereira (IBMC, Porto, Portugal) for helpful discussions. This work was supported by the Fundação para a Ciência e Tecnologia (Project no. POC/PI/41433/2001)

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Authors and Affiliations

  1. Instituto de Biologia Molecular e Celular Rua do Campo Alegre, 823, 4150-180, Porto, Portugal

    Lígia Lima, Ana Seabra, Paula Melo & Helena Carvalho

  2. Laboratoire des Interactions Plantes-Microorganismes INRA-CNRS, BP 27, 31326, Castanet-Tolosan Cedex, France

    Julie Cullimore

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  1. Lígia Lima
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  2. Ana Seabra
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  3. Paula Melo
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  4. Julie Cullimore
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  5. Helena Carvalho
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Correspondence to Helena Carvalho.

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Lima, L., Seabra, A., Melo, P. et al. Phosphorylation and subsequent interaction with 14-3-3 proteins regulate plastid glutamine synthetase in Medicago truncatula . Planta 223, 558–567 (2006). https://doi.org/10.1007/s00425-005-0097-8

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