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

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.

[1]  L. Heath,et al.  Increased expression of a 58-kDa protein kinase leads to changes in the CHO cell cycle. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Paul Nurse,et al.  Ordering S phase and M phase in the cell cycle , 1994, Cell.

[3]  Kim Nasmyth,et al.  The B-type cyclin kinase inhibitor p40 SIC1 controls the G1 to S transition in S. cerevisiae , 1994, Cell.

[4]  D. Bouchez,et al.  Normal differentiation patterns in plants lacking microtubular preprophase bands , 1995, Nature.

[5]  A. Shevchenko,et al.  Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. , 1996, Analytical chemistry.

[6]  D. Inzé,et al.  The Arabidopsis Cks1At protein binds the cyclin‐dependent kinases Cdc2aAt and Cdc2bAt , 1997, FEBS letters.

[7]  D. Inzé,et al.  Plant CDC2 is not only targeted to the pre‐prophase band, but also co‐localizes with the spindle, phragmoplast, and chromosomes , 1997, FEBS letters.

[8]  M. Kirschner,et al.  Stable transformation of an Arabidopsis cell suspension culture with firefly luciferase providing a cellular system for analysis of chaperone activity in vivo. , 1997, The Plant cell.

[9]  J. Peters,et al.  SCF and APC: the Yin and Yang of cell cycle regulated proteolysis. , 1998, Current opinion in cell biology.

[10]  D. N. Perkins,et al.  Probability‐based protein identification by searching sequence databases using mass spectrometry data , 1999, Electrophoresis.

[11]  S. Reed,et al.  Functional Characterization of Rpn3 Uncovers a Distinct 19S Proteasomal Subunit Requirement for Ubiquitin-Dependent Proteolysis of Cell Cycle Regulatory Proteins in Budding Yeast , 1999, Molecular and Cellular Biology.

[12]  B. Séraphin,et al.  A generic protein purification method for protein complex characterization and proteome exploration , 1999, Nature Biotechnology.

[13]  D. Inzé,et al.  Characterization of two distinct DP‐related genes from Arabidopsis thaliana 1 , 2000, FEBS letters.

[14]  I. Longden,et al.  EMBOSS: the European Molecular Biology Open Software Suite. , 2000, Trends in genetics : TIG.

[15]  Franky R. G. Terras,et al.  Functional Analysis of Cyclin-Dependent Kinase Inhibitors of Arabidopsis , 2001, The Plant Cell Online.

[16]  P. Genschik,et al.  Sub-cellular localisation of GFP-tagged tobacco mitotic cyclins during the cell cycle and after spindle checkpoint activation. , 2001, The Plant journal : for cell and molecular biology.

[17]  J R Yates,et al.  Selective degradation of ubiquitinated Sic1 by purified 26S proteasome yields active S phase cyclin-Cdk. , 2001, Molecular cell.

[18]  D. Inzé,et al.  Identification of novel cyclin-dependent kinases interacting with the CKS1 protein of Arabidopsis. , 2001, Journal of experimental botany.

[19]  D. Inzé,et al.  A Plant-specific Cyclin-dependent Kinase Is Involved in the Control of G2/M Progression in Plants* , 2001, The Journal of Biological Chemistry.

[20]  M. Menges,et al.  The Arabidopsis D-type Cyclins CycD2 and CycD3 Both Interact in Vivo with the PSTAIRE Cyclin-dependent Kinase Cdc2a but Are Differentially Controlled* , 2001, The Journal of Biological Chemistry.

[21]  Assen Roguev,et al.  Deciphering Protein Complexes and Protein Interaction Networks by Tandem Affinity Purification and Mass Spectrometry , 2002, Molecular & Cellular Proteomics.

[22]  Songwen Zhang,et al.  Interaction of p 58 PITSLRE , a G 2 / M-specific Protein Kinase , with Cyclin D 3 * , 2002 .

[23]  M. Menges,et al.  Synchronous Arabidopsis suspension cultures for analysis of cell-cycle gene activity. , 2002, The Plant journal : for cell and molecular biology.

[24]  Gary D Bader,et al.  Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry , 2002, Nature.

[25]  P. Bork,et al.  Functional organization of the yeast proteome by systematic analysis of protein complexes , 2002, Nature.

[26]  Songwen Zhang,et al.  Interaction of p58PITSLRE, a G2/M-specific Protein Kinase, with Cyclin D3* , 2002, The Journal of Biological Chemistry.

[27]  P. Shannon,et al.  Cytoscape: a software environment for integrated models of biomolecular interaction networks. , 2003, Genome research.

[28]  M. Umeda,et al.  Arabidopsis D-Type Cyclin CYCD4;1 Is a Novel Cyclin Partner of B2-Type Cyclin-Dependent Kinase1 , 2003, Plant Physiology.

[29]  P. Masson,et al.  Use of RNA Interference and Complementation To Study the Function of the Drosophila and Human 26S Proteasome Subunit S13 , 2003, Molecular and Cellular Biology.

[30]  M. Fromm,et al.  Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants. , 2004, The Plant journal : for cell and molecular biology.

[31]  M. Menges,et al.  Cryopreservation of Transformed and Wild-type Arabidopsis and Tobacco Cell Suspension Cultures , 2003 .

[32]  Giulio Superti-Furga,et al.  A physical and functional map of the human TNF-α/NF-κB signal transduction pathway , 2004, Nature Cell Biology.

[33]  D. Inzé,et al.  A technology platform for the fast production of monoclonal recombinant antibodies against plant proteins and peptides. , 2004, Journal of immunological methods.

[34]  Steven P Gygi,et al.  Large-scale characterization of HeLa cell nuclear phosphoproteins. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[35]  Brian T Chait,et al.  Targeted proteomic study of the cyclin-Cdk module. , 2004, Molecular cell.

[36]  M. Umeda,et al.  The Plant-Specific Kinase CDKF;1 Is Involved in Activating Phosphorylation of Cyclin-Dependent Kinase-Activating Kinases in Arabidopsis , 2004, The Plant Cell Online.

[37]  J. Murray,et al.  Differential stability of Arabidopsis D-type cyclins: CYCD3;1 is a highly unstable protein degraded by a proteasome-dependent mechanism. , 2004, The Plant journal : for cell and molecular biology.

[38]  A. Schnittger,et al.  Novel Functions of Plant Cyclin-Dependent Kinase Inhibitors, ICK1/KRP1, Can Act Non-Cell-Autonomously and Inhibit Entry into Mitosisw⃞ , 2005, The Plant Cell Online.

[39]  J. Bähler,et al.  Impairment of the TFIIH-associated CDK-activating kinase selectively affects cell cycle-regulated gene expression in fission yeast. , 2005, Molecular biology of the cell.

[40]  Martin Kuiper,et al.  BiNGO: a Cytoscape plugin to assess overrepresentation of Gene Ontology categories in Biological Networks , 2005, Bioinform..

[41]  M. Umeda,et al.  A distinct type of cyclin D, CYCD4;2, involved in the activation of cell division in Arabidopsis , 2006, Plant Cell Reports.

[42]  V. Rubio,et al.  An alternative tandem affinity purification strategy applied to Arabidopsis protein complex isolation. , 2005, The Plant journal : for cell and molecular biology.

[43]  P. Hilson,et al.  Modular cloning in plant cells. , 2005, Trends in plant science.

[44]  Wilhelm Gruissem,et al.  Global analysis of the core cell cycle regulators of Arabidopsis identifies novel genes, reveals multiple and highly specific profiles of expression and provides a coherent model for plant cell cycle control. , 2005, The Plant journal : for cell and molecular biology.

[45]  M. Montenarh,et al.  Cyclin H is targeted to the nucleus by C-terminal nuclear localization sequences , 2005, Cellular and Molecular Life Sciences.

[46]  Dirk Inzé,et al.  Cell cycle regulation in plant development. , 2006, Annual review of genetics.

[47]  Michael Gribskov,et al.  Protein-protein interactions of tandem affinity purification-tagged protein kinases in rice. , 2006, The Plant journal : for cell and molecular biology.

[48]  D. Inzé,et al.  SIAMESE, a Plant-Specific Cell Cycle Regulator, Controls Endoreplication Onset in Arabidopsis thaliana[W] , 2006, The Plant Cell Online.

[49]  M. Umeda,et al.  Diverse phosphoregulatory mechanisms controlling cyclin-dependent kinase-activating kinases in Arabidopsis. , 2006, The Plant journal : for cell and molecular biology.

[50]  T. Nakai,et al.  Arabidopsis KRPs have distinct inhibitory activity toward cyclin D2‐associated kinases, including plant‐specific B‐type cyclin‐dependent kinase , 2006, FEBS letters.

[51]  E. Nigg,et al.  A complex of two centrosomal proteins, CAP350 and FOP, cooperates with EB1 in microtubule anchoring. , 2005, Molecular biology of the cell.

[52]  M. Menges,et al.  Synchronization, transformation, and cryopreservation of suspension-cultured cells. , 2006, Methods in molecular biology.

[53]  Sean R. Collins,et al.  Global landscape of protein complexes in the yeast Saccharomyces cerevisiae , 2006, Nature.

[54]  P. Hilson.  Cloned sequence repertoires for small- and large-scale biology. , 2006, Trends in plant science.

[55]  P. Bork,et al.  Proteome survey reveals modularity of the yeast cell machinery , 2006, Nature.