title: The hairpin region of Ndc80 is important for the kinetochore recruitment of Mph1/MPS1 in fission yeast.
abstract: The establishment of proper kinetochore-microtubule attachments facilitates faithful chromosome segregation. Incorrect attachments activate the spindle assembly checkpoint (SAC), which blocks anaphase onset via recruitment of a cohort of SAC components (Mph1/MPS1, Mad1, Mad2, Mad3/BubR1, Bub1 and Bub3) to kinetochores. KNL1, a component of the outer kinetochore KMN network (KNL1/Mis12 complex/Ndc80 complex), acts as a platform for Bub1 and Bub3 localization upon its phosphorylation by Mph1/MPS1. The Ndc80 protein, a major microtubule-binding site, is critical for MPS1 localization to the kinetochores in mammalian cells. Here we characterized the newly isolated mutant ndc80-AK01 in fission yeast, which contains a single point mutation within the hairpin region. This hairpin connects the preceding calponin-homology domain with the coiled-coil region. ndc80-AK01 was hypersensitive to microtubule depolymerizing reagents with no apparent growth defects without drugs. Subsequent analyses indicated that ndc80-AK01 is defective in SAC signaling, as mutant cells proceeded into lethal cell division in the absence of microtubules. Under mitotic arrest conditions, all SAC components (Ark1/Aurora B, Mph1, Bub1, Bub3, Mad3, Mad2 and Mad1) did not localize to the kinetochore. Further genetic analyses indicated that the Ndc80 hairpin region might act as a platform for the kinetochore recruitment of Mph1, which is one of the most upstream SAC components in the hierarchy. Intriguingly, artificial tethering of Mph1 to the kinetochore fully restored checkpoint signaling in ndc80-AK01 cells, further substantiating the notion that Ndc80 is a kinetochore platform for Mph1. The hairpin region of Ndc80, therefore, plays a critical role in kinetochore recruitment of Mph1.
authors.affiliation: a The Francis Crick Institute, Lincoln's Inn Fields , London , United Kingdom.
authors.forename: Aldona Ewa
authors.initials: AE
authors.lastname: Chmielewska
authors.affiliation: a The Francis Crick Institute, Lincoln's Inn Fields , London , United Kingdom.
authors.forename: Ngang Heok
authors.initials: NH
authors.lastname: Tang
authors.affiliation: a The Francis Crick Institute, Lincoln's Inn Fields , London , United Kingdom.|b Hiroshima Research Center for Healthy Aging (HiHA), Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University , Higashi-Hiroshima , Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Toda
mesh_terms: D020816:Amino Acid Motifs; D018386:Kinetochores / Q000201:enzymology*; D059566:M Phase Cell Cycle Checkpoints; D008938:Mitosis; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport; D012568:Schizosaccharomyces / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000378:metabolism*; D050257:Tubulin Modulators / Q000494:pharmacology
chemical_list: D029702:Schizosaccharomyces pombe Proteins; D050257:Tubulin Modulators; C570446:Mph1 protein, S pombe; D011494:Protein Kinases
keywords: Mph1/MPS1; Ndc80; fission yeast; kinetochore; spindle assembly checkpoint