The kinetochore module Okp1 CENP‐Q /Ame1 CENP‐U is a reader for N‐terminal modifications on the centromeric histone Cse4 CENP‐A
2018; Springer Nature; Volume: 38; Issue: 1 Linguagem: Inglês
10.15252/embj.201898991
ISSN1460-2075
AutoresE. A. Anedchenko, Anke Samel‐Pommerencke, Tra My Tran Nguyen, Sara Shahnejat‐Bushehri, Juliane Pöpsel, Daniel Lauster, Andreas Herrmann, Juri Rappsilber, Alessandro Cuomo, Tiziana Bonaldi, Ann E. Ehrenhofer‐Murray,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle2 November 2018free access Source DataTransparent process The kinetochore module Okp1CENP-Q/Ame1CENP-U is a reader for N-terminal modifications on the centromeric histone Cse4CENP-A Ekaterina A Anedchenko Department of Molecular Cell Biology, Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany Search for more papers by this author Anke Samel-Pommerencke Department of Molecular Cell Biology, Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany Search for more papers by this author Tra My Tran Nguyen Department of Molecular Cell Biology, Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany Search for more papers by this author Sara Shahnejat-Bushehri Department of Molecular Cell Biology, Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany Search for more papers by this author Juliane Pöpsel Department of Molecular Cell Biology, Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany Search for more papers by this author Daniel Lauster Department of Experimental Biophysics, Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany Search for more papers by this author Andreas Herrmann Department of Experimental Biophysics, Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany Search for more papers by this author Juri Rappsilber orcid.org/0000-0001-5999-1310 Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Department of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany Search for more papers by this author Alessandro Cuomo Department of Experimental Oncology, European Institute of Oncology, Milan, Italy Search for more papers by this author Tiziana Bonaldi orcid.org/0000-0003-3556-1265 Department of Experimental Oncology, European Institute of Oncology, Milan, Italy Search for more papers by this author Ann E Ehrenhofer-Murray Corresponding Author [email protected] orcid.org/0000-0001-8709-1942 Department of Molecular Cell Biology, Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany Search for more papers by this author Ekaterina A Anedchenko Department of Molecular Cell Biology, Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany Search for more papers by this author Anke Samel-Pommerencke Department of Molecular Cell Biology, Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany Search for more papers by this author Tra My Tran Nguyen Department of Molecular Cell Biology, Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany Search for more papers by this author Sara Shahnejat-Bushehri Department of Molecular Cell Biology, Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany Search for more papers by this author Juliane Pöpsel Department of Molecular Cell Biology, Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany Search for more papers by this author Daniel Lauster Department of Experimental Biophysics, Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany Search for more papers by this author Andreas Herrmann Department of Experimental Biophysics, Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany Search for more papers by this author Juri Rappsilber orcid.org/0000-0001-5999-1310 Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Department of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany Search for more papers by this author Alessandro Cuomo Department of Experimental Oncology, European Institute of Oncology, Milan, Italy Search for more papers by this author Tiziana Bonaldi orcid.org/0000-0003-3556-1265 Department of Experimental Oncology, European Institute of Oncology, Milan, Italy Search for more papers by this author Ann E Ehrenhofer-Murray Corresponding Author [email protected] orcid.org/0000-0001-8709-1942 Department of Molecular Cell Biology, Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany Search for more papers by this author Author Information Ekaterina A Anedchenko1,‡, Anke Samel-Pommerencke1,‡, Tra My Tran Nguyen1, Sara Shahnejat-Bushehri1, Juliane Pöpsel1, Daniel Lauster2, Andreas Herrmann2, Juri Rappsilber3,4, Alessandro Cuomo5, Tiziana Bonaldi5 and Ann E Ehrenhofer-Murray *,1 1Department of Molecular Cell Biology, Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany 2Department of Experimental Biophysics, Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany 3Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK 4Department of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany 5Department of Experimental Oncology, European Institute of Oncology, Milan, Italy ‡These authors contributed equally to this work *Corresponding author. Tel: +49 30 2093 49630; E-mail: [email protected] EMBO J (2019)38:e98991https://doi.org/10.15252/embj.201898991 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Kinetochores are supramolecular assemblies that link centromeres to microtubules for sister chromatid segregation in mitosis. For this, the inner kinetochore CCAN/Ctf19 complex binds to centromeric chromatin containing the histone variant CENP-A, but whether the interaction of kinetochore components to centromeric nucleosomes is regulated by posttranslational modifications is unknown. Here, we investigated how methylation of arginine 37 (R37Me) and acetylation of lysine 49 (K49Ac) on the CENP-A homolog Cse4 from Saccharomyces cerevisiae regulate molecular interactions at the inner kinetochore. Importantly, we found that the Cse4 N-terminus binds with high affinity to the Ctf19 complex subassembly Okp1/Ame1 (CENP-Q/CENP-U in higher eukaryotes), and that this interaction is inhibited by R37Me and K49Ac modification on Cse4. In vivo defects in cse4-R37A were suppressed by mutations in OKP1 and AME1, and biochemical analysis of a mutant version of Okp1 showed increased affinity for Cse4. Altogether, our results demonstrate that the Okp1/Ame1 heterodimer is a reader module for posttranslational modifications on Cse4, thereby targeting the yeast CCAN complex to centromeric chromatin. Synopsis The conserved inner kinetochore proteins Okp1 and Ame1 are directly targeted to the centromeric histone H3 variant Cse4 in budding yeast, functioning as readers for post-translational modifications in its N-terminal tail. Saccharomyces cerevisiae Cse4CENP-A carries R37 methylation (R37Me) and K49 acetylation (K49Ac) in its extended N-terminal domain. The Cse4 N-terminus (Cse4N) interacts with Okp1CENP-Q/Ame1CENP-U, components of the Ctf19/CCAN kinetochore complex. R37Me and K49Ac in Cse4N reduce the interaction between Cse4N and Okp1/Ame1. Mutations in OKP1 and AME1 suppress the centromeric defects caused by mutation of Cse4-R37, and increase the binding affinity of Okp1/Ame1 to Cse4N. Introduction The proper segregation of chromosomes during mitosis is essential for the faithful transmission of genetic information from the mother to the daughter cell, and errors in this process contribute to carcinogenesis and sterility in humans. For segregation, kinetochores are assembled at the centromeres of the chromosomes and provide the contact to the microtubules that pull the sister chromatids apart during mitosis (Musacchio & Desai, 2017). Centromeric chromatin is defined by the presence of the histone H3 variant CENP-A (centromere protein A; Earnshaw & Migeon, 1985), which replaces canonical H3 at centromeric nucleosomes that are interspersed with H3-containing nucleosomes in higher eukaryotes (Bodor et al, 2014). The yeast Saccharomyces cerevisiae possesses a point centromere with a single nucleosome containing the CENP-A homolog Cse4, around which ~125 bp of centromeric DNA (CEN) are wrapped (Stoler et al, 1995; Meluh et al, 1998). Cse4 contains a histone fold domain that is homologous to CENP-A in other organisms. In addition, it has an extended N-terminal domain of 135 amino acids that is specific to closely related fungi and is essential for centromere function (Keith et al, 1999). The yeast kinetochore serves as a model for kinetochores from higher eukaryotes, which are thought to consist of modular repeats of this kinetochore unit (Westermann & Schleiffer, 2013). The core kinetochore is a large multiprotein complex consisting of ~30 unique proteins in S. cerevisiae (Musacchio & Desai, 2017) that are assembled in modules, several of which are present in multiple copies (Joglekar et al, 2009). At the outer kinetochore, the KMN network provides the contact to the microtubule (Fig 1; Varma & Salmon, 2012). Here, several copies of the Ndc80 complex (Wigge & Kilmartin, 2001), which is a dumbbell-shaped structure, interact with the Dam1 ring (Nogales & Ramey, 2009). This ring encircles the microtubule and links the kinetochore to the mitotic spindle. At its centromere-proximal side, the Ndc80 complex contacts the MIND complex (Kudalkar et al, 2015). Figure 1. Overview of proteins of the yeast kinetochoreThe Cse4-containing nucleosome is depicted with the extended N-terminus, which carries R37Me and K49Ac. COMA subunits are shown in colour. Download figure Download PowerPoint On the chromatin-proximal side of the kinetochore, the Ctf19 complex is an important component of the inner kinetochore that interacts with centromeric chromatin and serves as an assembly platform for the outer kinetochore (Foltz et al, 2006; Izuta et al, 2006; Okada et al, 2006). The Ctf19 complex is the yeast equivalent of the constitutive centromere-associated network (CCAN) in higher eukaryotes and contains 11 other proteins (Hornung et al, 2014). The COMA complex represents a subassembly within the Ctf19 complex, and it links the inner kinetochore to the MIND complex (De Wulf et al, 2003). COMA was named for the S. cerevisiae components Ctf19CENP-P (Hyland et al, 1999), Okp1CENP-Q (Ortiz et al, 1999), Mcm21CENP-O (Poddar et al, 1999) and Ame1CENP-U (Burns et al, 1994; Cheeseman et al, 2002; Gavin et al, 2002; superscript indicates the mammalian orthologs). The mammalian equivalent of yeast COMA is the CENP-O/P/Q/U complex (Musacchio & Desai, 2017). Ctf19 and Mcm21 both contain RWD domains and together form a Y-shaped heterodimer with flexible N-terminal extensions (Schmitzberger & Harrison, 2012). This heterodimer interacts with the Okp1/Ame1 heterodimer via Okp1 residues 317–343 (Schmitzberger et al, 2017). Loss of this contact results in the disruption of binding of two Ctf19 components, Chl4CENP-N and Iml3CENP-L, at the centromere. Next to its binding to Ctf19/Mcm21, Okp1/Ame1 furthermore forms a platform for interactions with other components of the Ctf19 complex, for instance Nkp1/Nkp2, as well as to Mif2CENP-C, which binds the Cse4-containing nucleosome (Kato et al, 2013; Falk et al, 2016; Xiao et al, 2017). Okp1/Ame1 also connects COMA to the outer kinetochore by interacting with the MIND complex (Hornung et al, 2014). Apart from the interaction with Mif2, little is known about how COMA is recruited to the centromeric nucleosome and how this may be regulated during the cell cycle. Ctf19 was shown to interact by two-hybrid analysis with Cse4 (Ortiz et al, 1999), but this has not been confirmed using biochemical methods. Furthermore, Okp1/Ame1 interacts with DNA, but the interaction is not specific to centromeric sequences (Hornung et al, 2014). Of note, the genes encoding Okp1, Ame1 and Mif2, but not other components of the Ctf19 complex, are essential for viability, implying that they perform non-redundant functions at the centromere. One possibility to regulate protein–protein interactions is by posttranslational modification (PTM). PTMs on histones are widely used to regulate chromatin function by altering interactions with chromatin-binding proteins ("reader modules"; Patel & Wang, 2013). Such readers include bromo- or YEATS domains for histone acetylation and chromo-, tudor- and PWWP domains for arginine or lysine methylation. However, this type of regulation has not been described for centromeric chromatin, and no reader modules are known within kinetochore proteins. Next to canonical histone modifications, PTMs have also been discovered in CENP-A homologs. In human cells, serine 7 (S7) in the N-terminus of CENP-A is phosphorylated by Aurora kinase during mitosis (Zeitlin et al, 2001; Kunitoku et al, 2003), and CENP-A is furthermore phosphorylated at S16 and S18 (Bailey et al, 2013). Also, the initiating methionine of CENP-A is cleaved, and the α-amino group of glycine 1 is trimethylated by NRMT1 (Sathyan et al, 2017). The absence of this modification reduces the recruitment of CCAN components to the centromere. Furthermore, several PTMs are known on Cse4 from S. cerevisiae. It is ubiquitylated by the ubiquitin ligase Psh1, which controls the levels of Cse4 in the cell and thus restricts its localization to centromeres (Hewawasam et al, 2010; Ranjitkar et al, 2010). In our earlier work, we identified the methylation of arginine 37 (R37Me), which lies in the essential N-terminal domain (END) of Cse4 (Keith et al, 1999), as a modification whose absence causes severe synthetic genetic interactions in combination with deletions or mutations in genes encoding proteins of the COMA subcomplex of the Ctf19 complex, arguing that mutation of this site disturbs the interaction of Cse4 with a kinetochore component (Samel et al, 2012). Cse4 is furthermore acetylated on lysine 49 and phosphorylated on S22, S33, S40 and S105 (Boeckmann et al, 2013). While Cse4-S33 phosphorylation plays a role in histone deposition at the centromere (Hoffmann et al, 2018), the functional role of the other PTMs has not been resolved. Here, we have investigated the molecular mechanism by which Cse4-R37 methylation and Cse4-K49 acetylation regulate the recruitment of the kinetochore to the centromere. Through genetic screens, we identified mutations in OKP1 (among others, okp1-R164C) and AME1 (among others, ame1-273*) that suppress synthetic growth defects of cse4-R37A. This suggested a) that Okp1/Ame1 binds the Cse4 N-terminus, and b) that Okp1/Ame1 binding to mutant Cse4 is restored by the suppressor mutations. In agreement with this, we found Okp1/Ame1 to form a stable complex in vitro with the Cse4 N-terminus, whereas no binding to Ctf19/Mcm21 was observed. Importantly, in vitro studies using microscale thermophoresis showed that the binding affinity of Cse4 to Okp1/Ame1 was reduced by R37 methylation and K49 acetylation, and this defect in binding to modified Cse4 was suppressed by Okp1-R164C. Altogether, our results demonstrate that Okp1/Ame1 is a reader module for the N-terminus of Cse4. It tethers the kinetochore to centromeric chromatin by binding to Cse4, and this interaction is inhibited by Cse4-R37 methylation and Cse4-K49 acetylation. Results Suppression of Cse4-R37 mutation by okp1-R164C Cse4 is methylated on R37 (Fig 2A), and mutation of this site (cse4-R37A) causes a synthetic growth defect in cells lacking Cbf1 (cbf1∆; Samel et al, 2012). Cbf1 binds the CDEI element of centromeric sequences (Cai & Davis, 1990). Posttranslational modifications on proteins can exert their function by regulating the interaction with another protein. We hypothesized that the growth defect of cbf1∆ cse4-R37A is caused by the abrogation of an interaction between Cse4 and an interaction partner, possibly a kinetochore protein. To identify this factor, we isolated mutations that suppress the temperature-sensitive growth defect of cbf1∆ cse4-R37A. We surmised that some of these mutations might improve the binding of the hypothesized factor to the mutant N-terminus of Cse4 and therefore restore centromere function. Several mutants were isolated, and the site of the extragenic suppressor was determined by whole-genome sequencing. Interestingly, we obtained one isolate that carried a mutation in OKP1, which encodes the COMA component Okp1 (Ortiz et al, 1999). The mutation causes the change of arginine 164 of Okp1 to cysteine (okp1-R164C), a residue that lies within the "core" region (amino acids 166–211) that is essential for viability (Schmitzberger et al, 2017). We constructed the okp1-R164C mutation de novo and tested its ability to suppress the temperature sensitivity of cbf1∆ cse4-R37A. Significantly, while cbf1∆ cse4-R37A cells showed a pronounced growth defect at elevated temperatures, this growth defect was suppressed by okp1-R164C (Fig 2B). A tentative biochemical interpretation of this genetic suppression is that Okp1 interacts with the N-terminus of Cse4, and that this interaction is regulated by methylation of R37. Figure 2. Mutations in OKP1 suppress growth defects of mutation of Cse4-R37 Overview of the amino acid sequence of Cse4. R37Me and K49Ac sites are part of the essential N-terminal domain (END, aa 28–60, yellow) and are indicated in red. The localization of α-helices in the histone fold domain is shown in grey. Amino acid residues that are relevant for this study are indicated with numbers. okp1-R164C suppressed the temperature-sensitive growth defect of cbf1∆ cse4-R37A. Serial dilutions of strains with the indicated genotypes were spotted on full medium and grown for 3 days at 30 or 37°C. The original okp1-R164C isolate from the suppressor screen is indicated as "UV mutagenesis". okp1-R164C suppressed the growth defect of cse4-R37A with chl4∆. Representation as in (B). okp1-R164C was unable to suppress the lethality of cse4-R37A with ame1-4. Tetrad dissection of a genetic cross between a cse4-R37A okp1-R164C and an ame1-4 strain is shown. The four spores from individual asci are aligned in vertical rows. okp1-R164C suppressed the maintenance defect of cse4-R37A for plasmids lacking the CDEI sequence of CEN6 (CEN ∆CDEI, at 37°C). Error bars give SD of at least three independent transformants. *P = 0.03. The okp1 mutations I45T, S94T and E208V suppressed the temperature-sensitive growth defect of cbf1∆ cse4-R37A. Strains with the indicated okp1 alleles on a plasmid (derivatives of AEY5584) are shown as in (B). Download figure Download PowerPoint We further determined whether other defects of cse4-R37A (Samel et al, 2012) were also suppressed by okp1-R164C. Indeed, okp1-R164C also suppressed the growth defect of cse4-R37A with mutations/deletions of other Ctf19 complex components (Fig 2C and D, Table 1). There was one notable exception to this, which is that the lethality of cse4-R37A with ame1-4 was not suppressed by okp1-R164C (Fig 2D). This observation is interesting in light of the fact that Okp1 and Ame1 are Ctf19 complex components that are essential for viability, and they form a heterodimer in vitro (Hornung et al, 2014). Table 1. Overview of the suppression of cse4-R37A phenotypes by okp1-R164C and ame1-273* Kinetochore component/complex Allele Synthetic phenotype with cse4-R37A (Samel et al, 2012) Suppression by okp1-R164C Suppression by by ame1-273* Cbf1 cbf1∆ Growth defect Suppression Suppression COMA ctf19∆ Lethality Partial suppression Partial suppression COMA mcm21∆ Lethality Partial suppression Partial suppression COMA ame1-4 Lethality No suppression n. d. COMA okp1-5 Growth defect n.d. No suppression Ctf19 complex iml3∆ Growth defect Suppression Suppression Ctf19 complex chl4∆ Growth defect Suppression Suppression Ctf19 complex ctf3∆ Growth defect Suppression Suppression n.d., not determined. cse4-R37A causes defects in chromosome and mini-chromosome segregation at centromeres that are compromised for CDEI function, which can be measured as an increased loss of plasmids that lack the CDEI sequence (Samel et al, 2012). Since okp1-R164C suppressed the temperature sensitivity of cbf1∆ cse4-R37A, we tested whether it also suppressed the plasmid maintenance defect of cse4-R37A. Indeed, while cse4-R37A caused an increased loss rate of a plasmid lacking CDEI (CEN ∆CDEI), this defect was decreased by additional okp1-R164C mutation (Fig 2E), showing that okp1-R164C suppressed the segregation defect of cse4-R37A at centromeres lacking CDEI function. cse4-R37A furthermore causes a cell-cycle arrest at the G2/M phase transition in cbf1∆ at the restrictive temperature (Samel et al, 2012), which reflects its defect in chromosome segregation. We therefore tested the effect of okp1-R164C on cell-cycle progression in cbf1∆ cse4-R37A by measuring the DNA content of cells by FACS analysis. Importantly, while cbf1∆ cse4-R37A cells showed an accumulation of cells with a 2n DNA content at the restrictive temperature, this arrest was partially suppressed in cbf1∆ cse4-R37A okp1-R164C (Fig EV1A), indicating that okp1-R164C partially restored centromere function and chromosome segregation to cse4-R37A. Click here to expand this figure. Figure EV1. okp1-R164C partially suppressed the G2/M arrest of cbf1Δ cse4-R37A at the restrictive temperature (related to Fig 2) A. WT (AEY4), cbf1Δ (AEY4816), cse4-R37A (AEY4965), cbf1Δ cse4-R37A (AEY4985), okp1-R164C (AEY5594) and cbf1Δ cse4-R37A okp1-R164C (AEY5974) cells were grown to early logarithmic phase at 23°C and shifted to 37°C for 5 h. DNA content of cells stained with SYTOX Green was measured by FACS analysis. B–D. Characterization of okp1 alleles. Alleles of okp1 that suppress cbf1∆ cse4-R37A defects had no defect with cbf1∆ (B), cse4-R37A (C) or in a wild-type background (D). Strains carrying the indicated alleles were serially diluted and grown on full medium at the indicated temperatures for 3 days. E. Mutation Okp1-R164 to alanine or glutamate did not suppress the temperature-sensitive defect of cbf1∆ cse4-R37A. Download figure Download PowerPoint We next asked whether other mutations in OKP1 could be identified that suppressed the defect of cbf1∆ cse4-R37A. Through a random mutagenesis of OKP1, two strong suppressor alleles, okp1-E208V and okp1-I45T, and one weaker suppressor, okp1-S94T, were isolated (Fig 2F). None of the okp1 alleles caused a growth defect on their own, nor in the presence of cbf1∆ or cse4-R37A alone (Fig EV1B–D). Okp1-E208, like R164, lies in the Okp1 core (166–211), the deletion of which is lethal, but is not required for the interaction with Ame1 (Schmitzberger et al, 2017), suggesting that it may be an interaction site with Cse4. Okp1-I45 and Okp1-S94 are in the flexible N-terminal region that is dispensable for binding to Ame1 and therefore may also represent a Cse4 binding region. We also tested substitutions of Okp1-R164 to alanine or glutamate, but both were unable to suppress the growth defect of cbf1∆ cse4-R37A (Fig EV1E), indicating that the mutation to cysteine bestowed special binding properties upon Okp1. Taken together, this showed that multiple alleles of OKP1 exist that suppress the defects of cse4-R37A and suggested that these alleles restore the hypothesized interaction of Okp1 with the Cse4 N-terminus. cse4-R37A reduces the recruitment of the MIND complex component Mtw1 to centromeric sequences under restrictive conditions (Samel et al, 2012). Since okp1-R164C suppressed the growth defect of cse4-R37A, we asked whether it also restored binding of Okp1 (Fig 3A) and Ame1 (Fig 3B) to the centromere as measured by chromatin immunoprecipitation (ChIP). Importantly, while the amount of Okp1 and Ame1 associated with CEN4 was reduced in cbf1∆ cse4-R37A cells grown at 37°C, their association was partially restored in okp1-R164C cells (Fig 3A and B). Furthermore, okp1-R164C restored the association of the MIND component Mtw1 to the centromere (Fig 3C). This indicated that the improved growth of the cells by the okp1 mutation was achieved by restoring the recruitment of other kinetochore components to the centromeric sequence. Figure 3. okp1-R164C restored binding of kinetochore components to centromeric sequences okp1-R164C restored binding of Okp1 to centromeric sequences. ChIP of 9xmyc-tagged Okp1 was performed in strains with the indicated genotypes that were grown at 37°C for 4 h before ChIP. Enrichment of CEN4 and POL1 (as a control) relative to input is given. Below, Western blot analysis of the amounts of 9xmyc-Okp1 and histone H2B (loading control) in whole cell extracts. okp1-R164C restored binding of Ame1 to centromeric sequences. ChIP of 9xmyc-tagged Ame1 was performed as in (A). Below, Western blot analysis of the amounts of 9xmyc-Ame1 and H2B as in (A). okp1-R164C restored binding of Mtw1 to centromeric sequences. ChIP of 9xmyc-Mtw1 and Western blots (right) are presented as in (A) and (B). okp1-R164C improved in vivo association of Okp1 with Cse4-R37A. Co-immunoprecipitation of Cse4-R37A and Okp1 was carried out in cells carrying cse4-R37A with (+) or without (−) 3xHA-tag and OKP1 or okp1-R164C with (+) or without (−) 9xmyc-tag (AEY5040, AEY5972, AEY5973, AEY6589, AEY6592). Cells were grown at 23°C or were shifted to 37°C for 5 h prior to harvesting. Cse4-R37A was immunoprecipitated using an α-HA antibody, and the presence of Cse4-R37A and Okp1 or Okp1-R164C in the immunoprecipitate was tested by Western blotting with α-HA and α-myc antibody, respectively. Left, inputs (the two bands indicate Okp1 and a shorter degradation product); right, α-HA immunoprecipitates. Data information: (A–C) Mean ± SD, n = 4 (P-values determined by Student's t-test). Source data are available online for this figure. Source Data for Figure 3 [embj201898991-sup-0007-SDataFig3.pdf] Download figure Download PowerPoint We next asked whether okp1-R164C affected the in vivo association of Okp1 and Cse4. To test this, co-immunoprecipitation (co-IP) was conducted between Cse4 and Okp1. Wild-type Okp1 was readily co-IPed with wt Cse4 (Fig EV2A). Upon IP of HA-tagged mutant Cse4-R37A, more Okp1-R164C than Okp1 was recovered in the immunoprecipitates (Fig 3D). We observed several bands for Okp1 (the lower bands most likely are proteolytic degradation products of Okp1). Altogether, we conclude that the in vivo association between Okp1 and Cse4-R37A (which could be direct or indirect) is improved by okp1-R164C. Click here to expand this figure. Figure EV2. Interaction between Cse4 and Okp1 (related to Figs 3D and 4) Co-immunoprecipitation of Okp1 and Cse4. Co-IP was carried out in cells carrying CSE4 with (+) or without (−) 3xHA-tag and OKP1 with (+) or without (−) 9xmyc-tag. Cells were grown at 30°C, and Cse4 was immunoprecipitated using an α-HA antibody (AEY2781, AEY5972, AEY6578). The presence of Cse4 and Okp1 in the immunoprecipitate was tested by Western blotting with α-HA and α-myc antibody, respectively. Left: inputs of the indicated strains; right: α-HA immunoprecipitates of the indicated strains. Two-hybrid analysis of the interaction between Cse4 (aa 11–139) and Okp1. Cells carrying the indicated constructs were streaked on minimal medium (+His) or on medium lacking histidine (−His) to test for activation of the two-hybrid reporter. Activity of the lacZ two-hybrid reporter of strains carrying the indicated two-hybrid constructs. Absence of in vitro interaction between Ctf19/Mcm21 and Cse4N. Recombinant Ctf19/Mcm21 and Cse4N were co-incubated prior to size-exclusion chromatography (SEC) as for Okp1/Ame1 in Fig 4A. Coomassie Blue-stained SDS–PAGE gels of SEC runs with Mcm21/Ctf19 (top), Cse4N (middle) and co-incubation of Ctf19/Mcm21 with Cse4N (bottom) are shown. Source data are available online for this figure. Download figure Download PowerPoint The Cse4 N-terminus binds Okp1/Ame1 The suppression of cse4-R37A defects by mutations in OKP1 suggests that cse4-R37A causes a loss of interaction of the Cse4 N-terminus with Okp1, and that the mutations in Okp1 restore binding of Okp1 to Cse4. We therefore sought to test whether there is a direct interaction between Okp1 and Cse4. To do so, we asked whether a recombinant N-terminal fragment of Cse4 (amino acids 21–129, Cse4N, Fig 2A) interacted in vitro with Okp1. Okp1 as obtained by recombinant expression in bacteria was only soluble upon co-expression with Ame1 (Hornung et al, 2014), and the two proteins formed a dimer in solution (Okp1/Ame1) as determined by size-exclusion chromatography (SEC, Fig 4A). Importantly, incubation of purified Okp1/Ame1 with recombinant Cse4N yielded a complex that eluted at higher molecular weight by SEC and contained all three proteins (Fig 4A). This demonstrated that Cse4N interacted in vitro with Okp1/Ame1, which was in line with our observation of a two-hybrid interaction between Cse4 and Okp1 (Fig EV2B and C). Figure 4. The N-terminus of Cse4 interacts in vitro with Okp1/Ame1 Cse4 (21–129, Cse4N) and Okp1/Ame1 were expressed and purified separately from bacteria and co-incubated before separation by analytical gel filtration. Top, representative size-exclusion chromatography (SEC) chromatogram with absorbance measurements at 280 nanometres (nm) is shown. Below, image of Coomassie Blue-stained SDS–PAGE gels with fractions from principal SEC peaks of runs with Okp1/Ame1 alone (top), Cse4N alone (middle) and Okp1/Ame1 preincubated with Cse4N before SEC (bottom). Molecular weight standards are shown in kilodalton (kDa). Intersubunit and self-link map for the Cse4N/Okp1/Ame1 comp
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