Human hsp90-sgt1 and skp1 form a conserved client-adaptor complex regulating global kinetochore structure through the balance of mis12 complex assembly and turnover

The kinetochore is a large multiprotein complex that links chromosomes to the mitotic spindle. Correct assembly ensures the faithful segregation of genetic material to resultant daughter cells during division. The task of assembling a multiprotein complex composed of 60-80 subunits, with high fidelity, represents a major cellular challenge. Failures of assembly reduce the affinity of microtubule-binding sites and impair the ability to monitor, integrate signals of, and correct chromosome mis-attachment. The consequences of this phenotype include chromosome breakage, mutation, and/or the inheritance of abnormal chromosome complements, which are associated with tumorigenesis. Understanding the mechanisms governing kinetochore assembly, regulation, and organization are therefore major issues in kinetochore biology.;Kinetochore assembly is best understood in the budding yeast Saccharomyces cerevisiae. In yeast a kinetochore keystone exists, the centromere binding factor 3 (CBF3), which is assembled by Hsp90, the co-chaperone Sgt1, and the SCF (Skp1-Cullin-F-Box) component Skp1. Assembly of the CBF3 is required for higher order kinetochore. Failure of CBF3 localization, either through CBF3 or Hsp90-Sgt1 loss of function mutants, result in kinetochore assembly defects and thus reduced mitotic fidelity. In contrast to the yeast kinetochore, which binds a single microtubule (i.e. a 'point' kinetochore), the mammalian kinetochore is multivalent and is composed of multiple kinetochore repeat structures -a 'regional' kinetochore. Because of the regional nature of the mammalian kinetochore, it forms an intricate network-like structure whereby proteins form many lateral attachments and outer and inner proteins influence each other's behavior. This level of complexity suggests that mechanisms must be in place to ensure proper formation and function of the kinetochore structure. Interestingly, the core regulatory subunits (Hsp90, Sgt1, and Skp1) involved in yeast CBF3 assembly are also conserved in mammalian cells and are required for kinetochore assembly. Hsp90 or Sgt1 loss of function results in aberrant kinetochore network formation resembling failed CBF3 assembly. However, mammalian cells do not contain a CBF3 homolog and the targets Hsp90-Sgt1 regulation are unknown.;To address this question we took a combined genetic and biochemical approach to identify the mammalian kinetochore targets of Hsp90-Sgt1. Here, we show that human Hsp90-Sgt1 interacts with the Mis12 complex, a so-called keystone complex required to assemble a large fraction of the kinetochore. Inhibition of Hsp90 or Sgt1 destabilizes the Mis12 complex and delays proper chromosome alignment due to inefficient formation of microtubule binding sites. Interestingly, co-inhibition of Sgt1 and the SCF subunit, Skp1, increases Mis12 complexes at kinetochores and restores timely chromosome alignment but forms less robust microtubule binding sites. These results suggest that Sgt1 functions to link Mis12 complexes to Hsp90 assembly and Skp1-directed turnover pathways. Consistent with this idea we find that Skp1 interacts directly with Mis12 complexes and that this interaction is important for Mis12 complex turnover. The interaction of Skp1 and Mis12 subunits is directed through a previously unidentified F-Box motif in Dsn1. Mutation of the Dsn1 F-box motif reduces Dsn1 turnover, leading to chromosome congression defects, and a reduction in the stability of kinetochore-microtubule contacts. We propose that Skp1 directed turnover of Dsn1 is required for maintain the fidelity of kinetochore-microtubule binding sites through the removal of non-functional Mis12 complexes. Taken together, these findings support a novel role for Hsp90-Sgt1 and Skp1 chaperones in ensuring the fidelity of multi-protein complexes through the balance of assembly and turnover.