title: Coupling unbiased mutagenesis to high-throughput DNA sequencing uncovers functional domains in the Ndc80 kinetochore protein of Saccharomyces cerevisiae.
abstract: During mitosis, kinetochores physically link chromosomes to the dynamic ends of spindle microtubules. This linkage depends on the Ndc80 complex, a conserved and essential microtubule-binding component of the kinetochore. As a member of the complex, the Ndc80 protein forms microtubule attachments through a calponin homology domain. Ndc80 is also required for recruiting other components to the kinetochore and responding to mitotic regulatory signals. While the calponin homology domain has been the focus of biochemical and structural characterization, the function of the remainder of Ndc80 is poorly understood. Here, we utilized a new approach that couples high-throughput sequencing to a saturating linker-scanning mutagenesis screen in Saccharomyces cerevisiae. We identified domains in previously uncharacterized regions of Ndc80 that are essential for its function in vivo. We show that a helical hairpin adjacent to the calponin homology domain influences microtubule binding by the complex. Furthermore, a mutation in this hairpin abolishes the ability of the Dam1 complex to strengthen microtubule attachments made by the Ndc80 complex. Finally, we defined a C-terminal segment of Ndc80 required for tetramerization of the Ndc80 complex in vivo. This unbiased mutagenesis approach can be generally applied to genes in S. cerevisiae to identify functional properties and domains.
authors.affiliation: Department of Biochemistry University of Washington, Seattle, Washington 98195.
authors.forename: Jerry F
authors.initials: JF
authors.lastname: Tien
authors.forename: Kimberly K
authors.initials: KK
authors.lastname: Fong
authors.forename: Neil T
authors.initials: NT
authors.lastname: Umbreit
authors.forename: Celia
authors.initials: C
authors.lastname: Payen
authors.forename: Alex
authors.initials: A
authors.lastname: Zelter
authors.forename: Charles L
authors.initials: CL
authors.lastname: Asbury
authors.forename: Maitreya J
authors.initials: MJ
authors.lastname: Dunham
authors.forename: Trisha N
authors.initials: TN
authors.lastname: Davis
mesh_terms: D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D055029:Inverted Repeat Sequences; D018386:Kinetochores / Q000737:chemistry / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA
chemical_list: D018797:Cell Cycle Proteins; C116003:DAM1 protein, S cerevisiae; D008869:Microtubule-Associated Proteins; C112472:NDC80 protein, S cerevisiae; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins
keywords: Hec1; Illumina; coiled coil; total internal reflection fluorescence (TIRF)