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Molecular & Cellular Proteomics 6:1226-1238, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

A Tandem Affinity Purification-based Technology Platform to Study the Cell Cycle Interactome in Arabidopsis thaliana^*,S

Jelle Van Leene^,,¶, Hilde Stals^,,¶, Dominique Eeckhout^,,¶, Geert Persiau^,, Eveline Van De Slijke^,, Gert Van Isterdael^,, Annelies De Clercq^,, Eric Bonnet^,, Kris Laukens^||,**, Noor Remmerie^||, Kim Henderickx^||, Thomas De Vijlder^||, Azmi Abdelkrim^||, Anne Pharazyn^||, Harry Van Onckelen^||, Dirk Inzé^,, Erwin Witters^||, and Geert De Jaeger^,,,

From the Department of Plant Systems Biology, Flanders Institute for Biotechnology and Department of Molecular Genetics, Ghent University, Technologiepark 927, B-9052 Gent, Belgium and ^|| Centre for Proteome Analysis and Mass Spectrometry, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerpen, Belgium

Defining protein complexes is critical to virtually all aspects of cell biology because many cellular processes are regulated by stable protein complexes, and their identification often provides insights into their function. We describe the development and application of a high throughput tandem affinity purification/mass spectrometry platform for cell suspension cultures to analyze cell cycle-related protein complexes in Arabidopsis thaliana. Elucidation of this protein-protein interaction network is essential to fully understand the functional differences between the highly redundant cyclin-dependent kinase/cyclin modules, which are generally accepted to play a central role in cell cycle control, in all eukaryotes. Cell suspension cultures were chosen because they provide an unlimited supply of protein extracts of actively dividing and undifferentiated cells, which is crucial for a systematic study of the cell cycle interactome in the absence of plant development. Here we report the mapping of a protein interaction network around six known core cell cycle proteins by an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, tandem affinity purification adapted for plant cells, matrix-assisted laser desorption ionization tandem mass spectrometry, data analysis, and functional assays. We identified 28 new molecular associations and confirmed 14 previously described interactions. This systemic approach provides new insights into the basic cell cycle control mechanisms and is generally applicable to other pathways in plants.

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