The structure of the Ctf19c/CCAN from budding yeast
- Harvard Medical School, United States
Abstract
Eukaryotic kinetochores connect spindle microtubules to chromosomal centromeres. A group of proteins called the Ctf19 complex (Ctf19c) in yeast and the constitutive centromere associated network (CCAN) in other organisms creates the foundation of a kinetochore. The Ctf19c/CCAN influences the timing of kinetochore assembly, sets its location by associating with a specialized nucleosome containing the histone H3 variant Cse4/CENP-A, and determines the organization of the microtubule attachment apparatus. We present here the structure of a reconstituted 13-subunit Ctf19c determined by cryo-electron microscopy at ~4 Å resolution. The structure accounts for known and inferred contacts with the Cse4 nucleosome and for an observed assembly hierarchy. We describe its implications for establishment of kinetochores and for their regulation by kinases throughout the cell cycle.
Data availability
We have deposited the model coordinates and cryo-EM maps in the PDB (6NUW) and EMDB (EMD-0523). Tracking files for imaging experiments are included as a source data file associated with Figure 3.
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Model coordinates from The structure of the Ctf19c/CCAN from budding yeastProtein Data Bank, 6NUW.
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Cryo-EM from The structure of the Ctf19c/CCAN from budding yeastElectron Microscopy Data Bank, EMD-0523.
Article and author information
Author details
Stephen C Harrison
Funding
Howard Hughes Medical Institute
- Stephen M Hinshaw
- Stephen C Harrison
Helen Hay Whitney Foundation
- Stephen M Hinshaw
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Hinshaw & Harrison
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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The structure of the Ctf19c/CCAN from budding yeast
eLife 8:e44239.
https://doi.org/10.7554/eLife.44239
Further reading
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- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
The two identical motor domains (heads) of dimeric kinesin-1 move in a hand-over-hand process along a microtubule, coordinating their ATPase cycles such that each ATP hydrolysis is tightly coupled to a step and enabling the motor to take many steps without dissociating. The neck linker, a structural element that connects the two heads, has been shown to be essential for head–head coordination; however, which kinetic step(s) in the chemomechanical cycle is ‘gated’ by the neck linker remains unresolved. Here, we employed pre-steady-state kinetics and single-molecule assays to investigate how the neck-linker conformation affects kinesin’s motility cycle. We show that the backward-pointing configuration of the neck linker in the front kinesin head confers higher affinity for microtubule, but does not change ATP binding and dissociation rates. In contrast, the forward-pointing configuration of the neck linker in the rear kinesin head decreases the ATP dissociation rate but has little effect on microtubule dissociation. In combination, these conformation-specific effects of the neck linker favor ATP hydrolysis and dissociation of the rear head prior to microtubule detachment of the front head, thereby providing a kinetic explanation for the coordinated walking mechanism of dimeric kinesin.
