HZwint-1 bridges the inner and outer kinetochore: Identification of the kinetochore localization domain and the hZw10-interaction domain

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 hHec l, hSpc24 and hMis12. As such, we proposed that hZwint-1 is a structural protein, part of the inner kinetochore scaffold and recruits hZwl0 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 hZwl0. 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.