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.
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.
Model coordinates from The structure of the Ctf19c/CCAN from budding yeastProtein Data Bank, 6NUW.
Cryo-EM from The structure of the Ctf19c/CCAN from budding yeastElectron Microscopy Data Bank, EMD-0523.
- Stephen M Hinshaw
- Stephen C Harrison
- Stephen M Hinshaw
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Andrea Musacchio, Max Planck Institute of Molecular Physiology, Germany
© 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.
Acid-sensing ion channels (ASICs) are trimeric proton-gated sodium channels. Recent work has shown that these channels play a role in necroptosis following prolonged acidic exposure like occurs in stroke. The C-terminus of ASIC1a is thought to mediate necroptotic cell death through interaction with receptor interacting serine threonine kinase 1 (RIPK1). This interaction is hypothesized to be inhibited at rest via an interaction between the C- and N-termini which blocks the RIPK1 binding site. Here, we use two transition metal ion FRET methods to investigate the conformational dynamics of the termini at neutral and acidic pH. We do not find evidence that the termini are close enough to be bound while the channel is at rest and find that the termini may modestly move closer together during acidification. At rest, the N-terminus adopts a conformation parallel to the membrane about 10 Å away. The distal end of the C-terminus may also spend time close to the membrane at rest. After acidification, the proximal portion of the N-terminus moves marginally closer to the membrane whereas the distal portion of the C-terminus swings away from the membrane. Together these data suggest that a new hypothesis for RIPK1 binding during stroke is needed.