title: Kinetochore biorientation in Saccharomyces cerevisiae requires a tightly folded conformation of the Ndc80 complex.
abstract: Accurate transmission of genetic material relies on the coupling of chromosomes to spindle microtubules by kinetochores. These linkages are regulated by the conserved Aurora B/Ipl1 kinase to ensure that sister chromatids are properly attached to spindle microtubules. Kinetochore-microtubule attachments require the essential Ndc80 complex, which contains two globular ends linked by large coiled-coil domains. In this study, we isolated a novel ndc80 mutant in Saccharomyces cerevisiae that contains mutations in the coiled-coil domain. This ndc80 mutant accumulates erroneous kinetochore-microtubule attachments, resulting in misalignment of kinetochores on the mitotic spindle. Genetic analyses with suppressors of the ndc80 mutant and in vitro cross-linking experiments suggest that the kinetochore misalignment in vivo stems from a defect in the ability of the Ndc80 complex to stably fold at a hinge in the coiled coil. Previous studies proposed that the Ndc80 complex can exist in multiple conformations: elongated during metaphase and bent during anaphase. However, the distinct functions of individual conformations in vivo are unknown. Here, our analysis revealed a tightly folded conformation of the Ndc80 complex that is likely required early in mitosis. This conformation is mediated by a direct, intracomplex interaction and involves a greater degree of folding than the bent form of the complex at anaphase. Furthermore, our results suggest that this conformation is functionally important in vivo for efficient error correction by Aurora B/Ipl1 and, consequently, to ensure proper kinetochore alignment early in mitosis.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195.
authors.forename: Jerry F
authors.initials: JF
authors.lastname: Tien
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195.
authors.forename: Neil T
authors.initials: NT
authors.lastname: Umbreit
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195.
authors.forename: Alex
authors.initials: A
authors.lastname: Zelter
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195 Department of Genome Sciences, University of Washington, Seattle, Washington 98195.
authors.forename: Michael
authors.initials: M
authors.lastname: Riffle
authors.affiliation: Institute for Systems Biology, Seattle, Washington 98109.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Hoopmann
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington 98195.
authors.forename: Richard S
authors.initials: RS
authors.lastname: Johnson
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037.
authors.forename: Bryan R
authors.initials: BR
authors.lastname: Fonslow
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037.
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington 98195.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: MacCoss
authors.affiliation: Institute for Systems Biology, Seattle, Washington 98109.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Moritz
authors.affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195.
authors.forename: Charles L
authors.initials: CL
authors.lastname: Asbury
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195 tdavis@uw.edu.
authors.forename: Trisha N
authors.initials: TN
authors.lastname: Davis
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D059447:Cell Cycle Checkpoints / Q000235:genetics; D018386:Kinetochores / Q000737:chemistry / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: C112472:NDC80 protein, S cerevisiae; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins
keywords: Hec1; linker scanning; mass spectrometry; optical trap; syntelic