title: hZwint-1 bridges the inner and outer kinetochore: identification of the kinetochore localization domain and the hZw10-interaction domain.
abstract: Accurate chromosome segregation in mitosis is required to maintain genetic stability. hZwint-1 [human Zw10 (Zeste white 10)-interacting protein 1] is a kinetochore protein known to interact with the kinetochore checkpoint protein hZw10. hZw10, along with its partners Rod (Roughdeal) and hZwilch, form a complex which recruits dynein-dynactin and Mad1-Mad2 complexes to the kinetochore and are essential components of the mitotic checkpoint. hZwint-1 localizes to the kinetochore in prophase, before hZw10 localization, and remains at the kinetochore until anaphase, after hZw10 has dissociated. This difference in localization timing may reflect a role for hZwint-1 as a structural kinetochore protein. In addition to hZw10, we have found that hZwint-1 interacts with components of the conserved Ndc80 and Mis12 complexes in yeast two-hybrid and GST (glutathione transferase) pull-down assays. Furthermore, hZwint-1 was found to have stable FRAP (fluorescence recovery after photobleaching) dynamics similar to hHec1, hSpc24 and hMis12. As such, we proposed that hZwint-1 is a structural protein, part of the inner kinetochore scaffold and recruits hZw10 to the kinetochore. To test this, we performed mutagenesis-based domain mapping to determine which regions of hZwint-1 are necessary for kinetochore localization and which are required for interaction with hZw10. hZwint-1 localizes to the kinetochore through the N-terminal region and interacts with hZw10 through the C-terminal coiled-coil domain. The two domains are at opposite ends of the protein as expected for a protein that bridges the inner and outer kinetochore.
authors.affiliation: Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Larissa J
authors.initials: LJ
authors.lastname: Vos
authors.forename: Jakub K
authors.initials: JK
authors.lastname: Famulski
authors.forename: Gordon K T
authors.initials: GK
authors.lastname: Chan
mesh_terms: D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020090:Chromosome Segregation; D003598:Cytoskeletal Proteins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018386:Kinetochores / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D008941:Spindle Apparatus
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D003598:Cytoskeletal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins; C120529:NDC80 protein, human; D009687:Nuclear Proteins; C484695:SPC24 protein, human; C484694:SPC25 protein, human; C488261:ZW10 protein, human; C413695:ZWINT protein, human