Structural requirements and dynamics of mitosin-kinetochore interaction in M phase.
Mitosin is a 350-kDa human nuclear protein which transiently associates with centromeres and spindle poles in M phase. Ultrastructure studies reveal that it is located at the outer kinetochore plate. In this work, we explored the detailed structural basis and dynamics of the mitosin-kinetochore interaction. Two major regions important for targeting to centromeres were identified by analyzing different deletion mutants expressed in CHO cells: (i) the "core region" between amino acids 2792 and 2887, which was essential for the centromere localization of mitosin; and (ii) the internal repeats between residues 2094 and 2487, which cooperated with the core region to achieve strong mitosin-kinetochore interaction. The core region is characteristic of two leucine zipper motifs. Deletion of either motif abolished the centromere localization activity. In addition, Cys2864, adjacent to the second motif, was also essential for the activity of the core region. In contrast, the internal repeats alone were insufficient for centromere localization. We propose that this region may serve as a regulatory domain to facilitate interaction of the core region with the kinetochore. We showed that mitosin molecules entering nuclei after nuclear envelope breakdown (NEBD) were not assembled onto kinetochores efficiently, suggesting that the mitosin-kinetochore interaction is stabilized prior to NEBD. This result supports the idea of an ordered process for kinetochore assembly. Our data also suggest that mitosin might interact with chromatin in interphase. Evidence for coordinated regulation between the centromere-targeting and the putative chromatin-binding activities is also provided.
Zhu
X
X
Shanghai Research Center of Life Sciences, Chinese Academy of Sciences, Shanghai 200031, China. xlzhu@iris.shlc.ac.cn
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D002503:Centromere / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D007399:Interphase / Q000502:physiology; D018386:Kinetochores / Q000378:metabolism*; D008840:Microfilament Proteins; D008938:Mitosis / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017384:Sequence Deletion; D014162:Transfection
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D017931:DNA Primers; D008840:Microfilament Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; C094772:centromere protein F; D004247:DNA