Abstract
The kinetochore is the macromolecular complex that assembles onto centromeric DNA and orchestrates the segregation of duplicated chromosomes. More than 60 components make up the budding yeast kinetochore, including inner kinetochore proteins that bind to centromeric chromatin and outer proteins that directly interact with microtubules. However, little is known about how these components assemble into a functional kinetochore and whether there are quality control mechanisms that monitor kinetochore integrity. We previously developed a method to isolate kinetochore particles via purification of the conserved Dsn1 kinetochore protein. We find that the Mub1/Ubr2 ubiquitin ligase complex associates with kinetochore particles through the CENP-CMif2 protein. Although Mub1/Ubr2 are not stable kinetochore components in vivo, they regulate the levels of the conserved outer kinetochore protein Dsn1 via ubiquitylation. Strikingly, a deletion of Mub1/Ubr2 restores the levels and viability of a mutant Dsn1 protein, reminiscent of quality control systems that target aberrant proteins for degradation. Consistent with this, Mub1/Ubr2 help to maintain viability when kinetochores are defective. Together, our data identify a previously unknown regulatory mechanism for the conserved Dsn1 kinetochore protein. We propose that Mub1/Ubr2 are part of a quality control system that monitors kinetochore integrity, thus ensuring genomic stability.
Highlights
Accurate chromosome segregation is essential to avoid the aneuploidy that is associated with cancer and birth defects [1]
The association between Ubr2 and Dsn1 was abolished in the absence of Mub1 (Figure 1C), suggesting that Mub1 acts as an adaptor protein that recruits Ubr2 onto kinetochore particles
We show that the Mub1/Ubr2 E3 ligase complex regulates the levels of the conserved Dsn1 kinetochore protein via ubiquitin-mediated proteolysis
Summary
Accurate chromosome segregation is essential to avoid the aneuploidy that is associated with cancer and birth defects [1]. Segregation is directed by the kinetochore, the macromolecular protein complex that assembles onto the centromeric region of each chromosome and that interacts with spindle microtubules during mitosis and meiosis [2–5]. The inner part of the kinetochore binds to centromeric DNA, whereas the outer portion interacts with microtubules. Greater than 60 kinetochore components have been identified in the budding yeast kinetochore, it remains unclear how the individual proteins assemble onto the centromere to form the macromolecular kinetochore structure [6]. Components of the CCAN (constitutive centromere-associated network, e.g. CENP-C) closely associate with CENP-A [9,10]. These inner kinetochore components are essential for the assembly of the outer kinetochore [11–13]. The outer kinetochore possesses microtubule-binding activity mediated through the KNL1 and Ndc complexes in the KMN (KNL1, Mis, Ndc complexes) network [14]. The Mis complex does not directly bind to microtubules, it is important for the assembly of the KMN and may be a keystone to promote outer kinetochore assembly [14–
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