Architecture of the human ndc80-hec1 complex, a critical constituent of the outer kinetochore.
The Ndc80 complex is a constituent of the outer plate of the kinetochore and plays a critical role in establishing the stable kinetochore-microtubule interactions required for chromosome segregation in mitosis. The Ndc80 complex is evolutionarily conserved and contains the four subunits Spc24, Spc25, Nuf2, and Ndc80 (whose human homologue is called Hec1). All four subunits are predicted to contain globular domains and extensive coiled coil regions. To gain an insight into the organization of the human Ndc80 complex, we reconstituted it using recombinant methods. The hydrodynamic properties of the recombinant Ndc80 complex are identical to those of the endogenous HeLa cell complex and are consistent with a 1:1:1:1 stoichiometry of the four subunits and a very elongated shape. Two tight Hec1-Nuf2 and Spc24-Spc25 subcomplexes, each stabilized by a parallel heterodimeric coiled coil, maintain this organization. These subcomplexes tetramerize via an interaction of the C- and N-terminal portions of the Hec1-Nuf2 and Spc24-Spc25 coiled coils, respectively. The recombinant complex displays normal kinetochore localization upon injection in HeLa cells and is therefore a faithful copy of the endogenous Ndc80 complex.
Ciferri
Claudio
C
Department of Experimental Oncology, European Institute of Oncology, Via Ripamonti 435, 20141 Milan, Italy.
De Luca
Jennifer
J
Monzani
Silvia
S
Ferrari
Karin J
KJ
Ristic
Dejan
D
Wyman
Claire
C
Stark
Holger
H
Kilmartin
John
J
Salmon
Edward D
ED
Musacchio
Andrea
A
D018797:Cell Cycle Proteins; D002845:Chromatography; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D008869:Microtubule-Associated Proteins / Q000737:chemistry; D008938:Mitosis; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D008869:Microtubule-Associated Proteins; C120529:NDC80 protein, human; C490197:NUF2 protein, human; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C484695:SPC24 protein, human; C484694:SPC25 protein, human; D005982:Glutathione Transferase