title: A tandem affinity purification-based technology platform to study the cell cycle interactome in Arabidopsis thaliana.
abstract: 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.
authors.affiliation: Department of Plant Systems Biology, Flanders Institute for Biotechnology, B-9052 Gent, Belgium.
authors.forename: Jelle
authors.initials: J
authors.lastname: Van Leene
authors.forename: Hilde
authors.initials: H
authors.lastname: Stals
authors.forename: Dominique
authors.initials: D
authors.lastname: Eeckhout
authors.forename: Geert
authors.initials: G
authors.lastname: Persiau
authors.forename: Eveline
authors.initials: E
authors.lastname: Van De Slijke
authors.forename: Gert
authors.initials: G
authors.lastname: Van Isterdael
authors.forename: Annelies
authors.initials: A
authors.lastname: De Clercq
authors.forename: Eric
authors.initials: E
authors.lastname: Bonnet
authors.forename: Kris
authors.initials: K
authors.lastname: Laukens
authors.forename: Noor
authors.initials: N
authors.lastname: Remmerie
authors.forename: Kim
authors.initials: K
authors.lastname: Henderickx
authors.forename: Thomas
authors.initials: T
authors.lastname: De Vijlder
authors.forename: Azmi
authors.initials: A
authors.lastname: Abdelkrim
authors.forename: Anne
authors.initials: A
authors.lastname: Pharazyn
authors.forename: Harry
authors.initials: H
authors.lastname: Van Onckelen
authors.forename: Dirk
authors.initials: D
authors.lastname: Inzé
authors.forename: Erwin
authors.initials: E
authors.lastname: Witters
authors.forename: Geert
authors.initials: G
authors.lastname: De Jaeger
mesh_terms: D017360:Arabidopsis / Q000166:cytology / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002850:Chromatography, Gel / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D025941:Protein Interaction Mapping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: D029681:Arabidopsis Proteins; D018797:Cell Cycle Proteins