Quantitative fragmentome mapping reveals novel, domain-specific partners for the modular protein RepoMan (recruits PP1 onto mitotic chromatin at anaphase).
RepoMan is a protein phosphatase 1 (PP1) regulatory subunit that targets the phosphatase to key substrates throughout the cell cycle. Most work to date has focused on the mitotic roles of RepoMan/PP1, although equally important interphase role(s) have been demonstrated. Initial mapping of the interactome of nuclear RepoMan, both endogenous and tagged, was complicated by various factors, including antibody cross-reactivity and low sensitivity of the detection of chromatin-associated partners above the high background of proteins that bind nonspecifically to affinity matrices. We therefore adapted the powerful combination of fluorescence imaging with labeling-based quantitative proteomics to map the "fragmentomes" of specific regions of RepoMan. These regions demonstrate distinct localization patterns and turnover dynamics that reflect underlying binding events. The increased sensitivity and signal-to-noise ratio provided by this unique approach facilitated identification of a large number of novel RepoMan interactors, several of which were rigorously validated in follow-up experiments, including the association of RepoMan/PP1 with a specific PP2A-B56γ complex, interaction with ribosomal proteins and import factors involved in their nucleocytoplasmic transport and interaction with proteins involved in the response to DNA damage. This same strategy can be used to investigate the cellular roles of other modular proteins.
Department of Cellular and Molecular Medicine and Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Ontario, Canada.
Michèle
M
Prévost
Delphine
D
Chamousset
Isha
I
Nasa
Emily
E
Freele
Nick
N
Morrice
Greg
G
Moorhead
Laura
L
Trinkle-Mulcahy
D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D054648:Protein Phosphatase 2 / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D017476:Receptors, Neuropeptide Y / Q000378:metabolism
C509224:CDCA2 protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D010446:Peptide Fragments; D021122:Protein Subunits; D020543:Proteome; D017476:Receptors, Neuropeptide Y; C109491:neuropeptide Y4 receptor; D054648:Protein Phosphatase 2