title: Synthetic physical interactions map kinetochore regulators and regions sensitive to constitutive Cdc14 localization.
abstract: The location of proteins within eukaryotic cells is often critical for their function and relocation of proteins forms the mainstay of regulatory pathways. To assess the importance of protein location to cellular homeostasis, we have developed a methodology to systematically create binary physical interactions between a query protein and most other members of the proteome. This method allows us to rapidly assess which of the thousands of possible protein interactions modify a phenotype. As proof of principle we studied the kinetochore, a multiprotein assembly that links centromeres to the microtubules of the spindle during cell division. In budding yeast, the kinetochores from the 16 chromosomes cluster together to a single location within the nucleus. The many proteins that make up the kinetochore are regulated through ubiquitylation and phosphorylation. By systematically associating members of the proteome to the kinetochore, we determine which fusions affect its normal function. We identify a number of candidate kinetochore regulators, including the phosphatase Cdc14. We examine where within the kinetochore Cdc14 can act and show that the effect is limited to regions that correlate with known phosphorylation sites, demonstrating the importance of serine phospho-regulation for normal kinetochore homeostasis.
authors.affiliation: The Francis Crick Institute, Mill Hill Laboratory, London NW7 1AA, United Kingdom.
authors.forename: Guðjón
authors.initials: G
authors.lastname: Ólafsson
authors.affiliation: The Francis Crick Institute, Mill Hill Laboratory, London NW7 1AA, United Kingdom Peter.Thorpe@crick.ac.uk.
authors.forename: Peter H
authors.initials: PH
authors.lastname: Thorpe
mesh_terms: D000483:Alleles; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002455:Cell Division; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006706:Homeostasis; D018386:Kinetochores / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D009154:Mutation; D010641:Phenotype; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism; D040901:Proteomics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D008941:Spindle Apparatus
chemical_list: C074860:CDC14 protein, S cerevisiae; D018797:Cell Cycle Proteins; C120362:MTW1 protein, S cerevisiae; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D010744:Phosphoric Monoester Hydrolases; D017027:Protein Tyrosine Phosphatases
keywords: CDK; MIND; Mtw1; kinetochore; phosphatase