title: Protein purification technique that allows detection of sumoylation and ubiquitination of budding yeast kinetochore proteins Ndc10 and Ndc80.
abstract: Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.
authors.affiliation: Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institute of Health.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Ohkuni
authors.affiliation: Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institute of Health.
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Takahashi
authors.affiliation: Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institute of Health; basraim@mail.nih.gov.
authors.forename: Munira A
authors.initials: MA
authors.lastname: Basrai
mesh_terms: D015153:Blotting, Western; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D047468:Immunoprecipitation; D018386:Kinetochores / Q000378:metabolism*; D009687:Nuclear Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D058207:Sumoylation; D025801:Ubiquitin / Q000378:metabolism; D054875:Ubiquitination
chemical_list: C081071:CBF2 protein, S cerevisiae; D004268:DNA-Binding Proteins; C112472:NDC80 protein, S cerevisiae; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin