Abstract
The four-protein Ndc80 complex, an essential kinetochore component conserved from yeast to humans, plays an indispensable role in proper chromosome alignment and segregation during mitosis. In higher eukaryotes, the homologous complex probably resides in the middle domain of the trilaminar kinetochore, linking centromeric heterochromatin with microtubule-associated structures. We have prepared recombinant Ndc80 complex by pairwise coexpression of its components (Ndc80p and Nuf2p; Spc24p and Spc25p) and shown that they form independently stable subcomplexes. Rotary shadowing electron microscopy, combined with limited proteolysis and antibody labeling, demonstrates that the heterotetrameric Ndc80 complex is an approximately 570-A-long rod, with globular regions at either end. The shaft contains alpha-helical coiled-coil segments from each of the two subcomplexes, linked end-to-end. When integrated with published observations derived from inactivating the components of Ndc80, the molecular organization we deduce suggests that the Spc24p/Spc25p end of the rod faces the centromere and the Ndc80p/Nuf2p end faces a spindle microtubule.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Cell Line
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Centromere / metabolism
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Chromosomal Proteins, Non-Histone / chemistry
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Chromosomal Proteins, Non-Histone / genetics
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Chromosomal Proteins, Non-Histone / metabolism
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Chromosomal Proteins, Non-Histone / ultrastructure
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Kinetochores / chemistry*
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Microtubules / metabolism
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Multiprotein Complexes / chemistry
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Multiprotein Complexes / genetics
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Multiprotein Complexes / metabolism
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Multiprotein Complexes / ultrastructure
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Nuclear Proteins / chemistry*
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism
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Nuclear Proteins / ultrastructure*
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Protein Conformation
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Protein Structure, Secondary
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Protein Subunits / chemistry
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Protein Subunits / genetics
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Protein Subunits / metabolism
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Saccharomyces cerevisiae / chemistry*
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Saccharomyces cerevisiae Proteins / chemistry*
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism
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Saccharomyces cerevisiae Proteins / ultrastructure*
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Spodoptera
Substances
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Chromosomal Proteins, Non-Histone
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Multiprotein Complexes
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NDC80 protein, S cerevisiae
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NUF2 protein, S cerevisiae
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Nuclear Proteins
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Protein Subunits
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Saccharomyces cerevisiae Proteins
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Spc24 protein, S cerevisiae
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Spc25 protein, S cerevisiae