A comprehensive analysis of the 14-3-3 interactome in barley leaves using a complementary proteomics and two-hybrid approach.
This study describes the identification of over 150 target proteins of the five 14-3-3 isoforms in 7-d-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, for example, Serpin, RF2A, WPK4 kinase, P-type proton-translocating adenosine triphosphatase, 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-kinase domain-interacting protein 129. 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, a REPRESSION OF SHOOT GROWTH ortholog named RF2A was identified; these transcription factors play important roles in the abscisic acid and gibberellin 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.
Schoonheim
Peter J
PJ
Department of Structural Biology, Faculty of Earth and Life Sciences, Vrije Universiteit, 1081 HV Amsterdam, The Netherlands.
Veiga
Helena
H
Pereira
Daniel da Costa
Dda C
Friso
Giulia
G
van Wijk
Klaas J
KJ
de Boer
Albertus H
AH
D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D001467:Hordeum / Q000235:genetics / Q000378:metabolism*; D018515:Plant Leaves / Q000378:metabolism*; D020033:Protein Isoforms; D040901:Proteomics; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques
D048948:14-3-3 Proteins; D020033:Protein Isoforms