Plant Physiology Preview
Published on December 15, 2006; 10.1104/pp.106.090159
OPEN ACCESS ARTICLE
Received September 21, 2006
Accepted December 6, 2006
A Comprehensive Analysis of the 14-3-3 Interactome in Barley Leaves Using a Complementary Proteomics and Two-hybrid Approach
Peter J. Schoonheim , Helena Veiga , Daniel da Costa Pereira , Giulia Friso , Klaas J. van Wijk , and Albertus H. de Boer *
Department of Structural Biology, Faculty of Earth and Life Sciences, Vrije Universiteit, De Boelelaan, 1085, 1081 HV Amsterdam, The Netherlands; Department of Plant Biology, Cornell University, Emerson Hall 3rd Floor, Tower Rd, Ithaca, NY 14853, USA
* Corresponding author; email: Bert.de.boer{at}falw.vu.nl.
This study describes the identification of over 150 target proteins of the five 14-3-3 isoforms in 7 days old barley (Hordeum vulgare cv. Himalaya) seedlings using yeast two-hybrid screens complemented with 14-3-3 protein affinity purification and tandem mass spectrometry. Independent experiments for a subset of genes confirmed the yeast-two hybrid interactions, demonstrating a low false positive identification rate. These combined approaches resulted in the identification of more than 150 putative targets; 15 % were previously reported to be 14-3-3 interactors, including e.g. Serpin, RF2A, WPK4 kinase, P-type H+-ATPase, EF1A, glutamine synthetase, and invertases. The affinity purification resulted in 30 interactors of which 44 % function in metabolism, while the yeast two hybrid screens identified 132 different proteins with 35 % of the proteins involved in signal transduction. A number of proteins have a well described function in hormonal signaling, such as the auxin transport protein PIN1 and NPH3 and components of the brassinosteroid pathway, such as the receptor kinase BAK1 (OsPERK1) and BRI1-KD interacting protein 129 (BIP129). However, 14-3-3 interactions with these signal mediators have not been confirmed in the affinity purification. Confirmations of the 14-3-3 interaction with the three ABF-like transcription factors are shown using far western analysis. Also, an RSG orthologue named RF2A were identified; these transcription factors play important roles in the ABA and GA pathways, respectively. We speculate that 14-3-3 proteins have a role in cross-talk between these hormonal pathways. The specificity and complementary nature of both the affinity purification and the yeast two-hybrid approaches is discussed.
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