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Hsp90–Sgt1 and Skp1 Target Human Mis12 Complexes to Ensure Efficient Formation of Kinetochore–Microtubule Binding Sites

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Hsp90–Sgt1 and Skp1 Target Human Mis12 Complexes to Ensure Efficient Formation of Kinetochore–Microtubule Binding Sites Published April 19, 2010 JCB: Article Hsp90–Sgt1 and Skp1 target human Mis12 complexes to ensure efficient formation of kinetochore–microtubule binding sites Alexander E. Davies and Kenneth B. Kaplan The Section of Molecular and Cellular Biology, University of California, Davis, Davis, CA 95616 he formation of functional kinetochores requires the due to inefficient formation of microtubule-binding sites. accurate assembly of a large number of protein Interestingly, coinhibition of Sgt1 and the SCF subunit, T complexes. The Hsp90–Sgt1 chaperone complex is Skp1, increases Mis12 complexes at kinetochores and re- important for this process; however, its targets are not stores timely chromosome alignment but forms less-robust conserved and its exact contribution to kinetochore as- microtubule-binding sites. We propose that a balance of sembly is unclear. Here, we show that human Hsp90–Sgt1 Mis12 complex assembly and turnover is required for the interacts with the Mis12 complex, a so-called keystone efficient and accurate assembly of kinetochore–microtubule Downloaded from complex required to assemble a large fraction of the binding sites. These findings support a novel role for Hsp90– kinetochore. Inhibition of Hsp90 or Sgt1 destabilizes the Sgt1 chaperones in ensuring the fidelity of multiprotein Mis12 complex and delays proper chromosome alignment complex assembly. on October 3, 2016 Introduction Kinetochores are assembled from multiple distinct protein com- few mutants completely eliminate kinetochore–microtubule bind- plexes to form a multivalent microtubule-binding site. Kineto- ing in yeast, which suggests that a strict hierarchy between chores contact the lateral faces of microtubules, and these initial centromeric DNA and microtubule binding may be an over- low-affinity contacts are believed to mature to more stable end- simplification. CBF3 is composed of three core subunits, and on attachments (for a recent review see Cheeseman and Desai, the Hsp90–Sgt1 chaperone complex balances its assembly with 2008). A combination of microtubule- and motor-derived forces its turnover (Rodrigo-Brenni et al., 2004). Analysis of recon- acting at the kinetochore and along chromosome arms contribute stituted Hsp90–Sgt1 complexes led to the proposed model that to chromosome alignment and establish tension between sister Sgt1 acts as an adapter, linking Hsp90 to a subset of its clients chromatids during metaphase (Hays and Salmon, 1990; Sharp (Catlett and Kaplan, 2006). Sgt1 directly interacts with Hsp90 THE JOURNAL OF CELL BIOLOGY et al., 2000). The identification of a large number of kinetochore and with Skp1, a protein shared with both CBF3 and the SCF proteins from multiple species has greatly advanced our under- E3 ubiquitin ligase complex. The SCF-dependent turnover of standing of kinetochore structure, yet the rules for assembling CBF3 subunits suggests that Sgt1 connects Hsp90 clients to these complexes are still poorly understood. SCF-mediated degradation (Kaplan et al., 1997). Consistent In budding yeast, the centromere binding complex 3 with this idea, mutants that affect the affinity of Sgt1 for Skp1 (CBF3) is first assembled before it can bind to sequence- favor either assembly or turnover of CBF3 (Rodrigo-Brenni specific elements on centromeric DNA (CEN-DNA) and recruit et al., 2004). additional complexes that form a single microtubule-binding In contrast to the relatively simple point kinetochores site (Espelin et al., 1997; Kaplan et al., 1997). After CBF3 that form on sequence-determined centromeres in budding binds to CEN-DNA, the assembly of a functional kinetochore yeast, specification of mammalian kinetochores is largely epi- is thought to occur in a hierarchical manner, with complexes genetic and is dependent on the proper loading of nucleosomes building outwards until a microtubule-binding site forms (Ortiz et al., 1999). However, it is interesting to note that relatively © 2010 Davies and Kaplan This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub- Correspondence to Kenneth B. Kaplan: [email protected] lication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, Abbreviation used in this paper: ACA, anti-centromere antigen. as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). Supplemental Material can be found at: /content/suppl/2010/04/18/jcb.200910036.DC1.html The Rockefeller University Press $30.00 J. Cell Biol. Vol. 189 No. 2 261–274 www.jcb.org/cgi/doi/10.1083/jcb.200910036 JCB 261 Published April 19, 2010 containing the histone H3 variant CENP-A (for a review see of high-affinity microtubule-binding sites after Sgt1 inhibition. Gieni et al., 2008). The CENP-A nucleosome is required to recruit Interestingly, inhibition of Skp1 reduces the robustness of micro- a kinetochore assembly platform in interphase that consists of tubule attachment sites, arguing that both assembly and turn- the CENP-H, CENP-M, and CENP-O complexes, also called the over of Mis12 are important for proper kinetochore function. constitutive centromere complex, or CCAN (Okada et al., 2006; Cheeseman and Desai, 2008). Outer kinetochore complexes are Results assembled at centromeres in the G2 stage of the cell cycle (Hori et al., 2003; Cheeseman et al., 2004; Yang et al., 2008); Knl1 Hsp90 is required for Dsn1 stability and and the Mis12 complex are believed to modulate the forma- Mis12 complex localization tion of the Ndc80 microtubule-binding complex (Cheeseman Previous studies using Hsp90 inhibitors and Sgt1 siRNA et al., 2006). Rather than forming individual microtubule-binding suggest that the chaperone complex is required for recruit- units, electron tomography experiments suggest that micro- ment of both constitutive and outer kinetochore complexes tubule-binding sites form a network that allows both low- and (Steensgaard et al., 2004; Niikura et al., 2006). However, be- high-affinity microtubule interactions (Dong et al., 2007). It has cause of the interdependence of kinetochore complexes during been suggested that this architecture allows for an initial low- assembly, it is unclear which kinetochore complex or com- affinity capture of microtubules by kinetochores followed by a plexes might require chaperone function. To address this issue, maturation process that gives rise to high-affinity interactions. we used the rapid kinetics of Hsp90 inhibition by 17-(allyl- Implicit in this model is the idea that the proper assembly of a amino)-17-demethoxygeldanamycin (17-AAG) to identify kinetochore network is necessary to ensure the efficient forma- kinetochore complexes most immediately affected. After inhi- tion of robust microtubule attachment sites. bition of Hsp90, clients often exhibit a decrease in protein levels, Experiments to test the interdependency of kinetochore either because they degrade or become insoluble. After treat- proteins on assembly of a microtubule-binding site are also con- ment of HeLa cells for 12–18 h with 100 nM 17-AAG, immuno- Downloaded from sistent with a network-like organization. For example, inhibi- blotting showed that both outer (e.g., Ndc80Hec1) and constitutive tion of the outer kinetochore protein Knl-1 reduces the formation kinetochore complexes (e.g., CENP-K, -H, -U, and -N) remain of microtubule attachment sites that include Ndc80Hec1 as well at control levels. In contrast, the Dsn1 subunit of the Mis12 as the “keystone” complex, Mis12 (Cheeseman et al., 2008). complex was reduced to 65% of control; the levels of other Similarly, the depletion of Mis12 subunits reduces the levels Mis12 subunits were unaffected by 17-AAG (Fig. 1 A). Treat- of both the outer kinetochore protein Ndc80Hec1 and the inner ment with 500 nM 17-AAG for 18 h further decreased the on October 3, 2016 kinetochore proteins CENP-H and CENP-A (Kline et al., 2006), levels of Dsn1 to less than half (47% of control; Fig. 1 B). which suggests that kinetochore complexes assemble into an Consistent with the immunoblots, short-term treatment of cells interdependent network. with 500 nM 17-AAG resulted in a general decrease in Mis12 Despite advances in understanding the dependencies of subunits at kinetochores (Fig. 1 C). It is interesting to note that kinetochore protein assembly at centromeres, it is less clear not all Mis12 components behaved the same way after 17-AAG how individual subunits correctly assemble into subcomplexes treatment; Dsn1 levels at kinetochores were decreased on aver- and then into higher-order structures. The fact that both simple age to 78% of control, whereas Nnf1 was more dramatically and complex kinetochores require Hsp90–Sgt1 suggests that reduced (63% of control; Fig. 1 D). The Nsl1 signal was simi- chaperones provide a conserved mechanism for assembling large larly decreased at kinetochores but was also found in large protein networks. Although mammalian cells lack a homologue aggregates near chromosomes (Fig. 1 C, arrow). The observed of CBF3, inhibition of either Hsp90 or Sgt1 leads to the re- heterogeneity suggests that Mis12 subunits have different fates duction in a large number of kinetochore-associated proteins in the absence of Hsp90. Overall, these observations support (Steensgaard et al., 2004; Niikura et al., 2006) and reduces the idea that the Mis12 complex, and in particular Dsn1, is a the efficiency of microtubule attachments. The partial loss target of the
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