Report |
Address correspondence to Georjana Barnes, Department of Molecular and Cell Biology, Barker Hall, University of California, Berkeley, Berkeley, CA 94720. Tel.: (510) 642-5962. Fax: (510) 643-0062. email: gbarnes{at}socrates.berkeley.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: microtubule; spindle; mitosis; nucleosome; Aurora kinase
The present address of I.M. Cheeseman is Ludwig Institute for Cancer Research University of California, San Diego, La Jolla, CA 92093-0660.
Abbreviations used in this paper: ChIP, chromatin immunoprecipitation; MAST, motif alignment and search tool; TAP, tandem affinity purification.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
A variety of strategies have been used to investigate the organization of the budding yeast kinetochore including two-hybrid analyses (Ortiz et al., 1999; Measday et al., 2002; Shang et al., 2003), biochemical purifications of kinetochore subcomplexes (Cheeseman et al., 2001, 2002b; Janke et al., 2001, 2002; Wigge and Kilmartin, 2001; Li et al., 2002), and chromatin immunoprecipitation (ChIP) analysis, which can establish the dependency requirements for the recruitment of a kinetochore protein to centromeric DNA (Meluh and Koshland, 1997; He et al., 2001; Measday et al., 2002; Pot et al., 2003).
We have described previously our purification of the kinetochore proteins that comprise the yeast central and outer kinetochore (Cheeseman et al., 2002b). Here, we examined the DNA binding components of the inner kinetochore. In addition to defining a new kinetochore subcomplex, this analysis has provided insights into how the various subcomplexes interact with each other and suggests a model for a higher order kinetochore structure, which has been conserved throughout evolution.
![]() |
Results and discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The essential CBF3 complex is a fundamental determinant of budding yeast kinetochore structure. In its absence all known kinetochore proteins fail to associate with centromeric DNA (for review see Cheeseman et al., 2002a). Interestingly, purification of the CBF3 complex using tagged Cep3p resulted in the recovery of the CBF3 subunits Ctf13 and Skp1p (Fig. 1), but not Ndc10p, which is present in the complex when it is isolated by DNA-affinity chromatography (Lechner and Carbon, 1991). Previous studies have demonstrated a distinct assembly pathway for the CBF3 complex with Ndc10p joining a Skp1pCtf13pCep3p subcomplex as the final assembly step (Russell et al., 1999). Although no other inner or central kinetochore proteins copurified with Cep3p, two proteins related to kinetochore function were detected in trace amounts. These proteins include Hir1p, which together with the chromatin assembly factor CAF-1 has a role in building functional kinetochores (Sharp et al., 2002), and Cdc4p, a subunit of the SCF ubiquitin ligase. The purification of Cbf1p, which binds to the CDEI centromeric element and shows limited homology to metazoan CENP-B, did not reveal associations with other kinetochore proteins under our experimental conditions.
|
The second set of proteins that copurifies with Mif2p consists of the kinetochore protein Mtw1p (Goshima and Yanagida, 2000) and the proteins Nnf1p, Dsn1p, and Nsl1p. Recently, a role in chromosome segregation has been demonstrated for the latter three proteins (Euskirchen, 2002). We also found that Nnf1p, Dsn1p, and Nsl1p specifically associate with centromeric DNA in ChIP experiments (Fig. S2, available at http://www.jcb.org/cgi/content/full/10.1083/jcb.200305100/DC1) establishing them as bona fide kinetochore components. Together, these data provide a physical basis for their genetic interactions with Mtw1p (Euskirchen, 2002). As judged by the relative band intensity on Coomassie-stained gels, Cse4p and the histones, as well as Mtw1p, Nnf1p, Dsn1p, and Nsl1p, all copurified substoichiometrically with Mif2p.
In addition to its associations with Mif2p, Mtw1p associates weakly with the Ctf19 complex (Cheeseman et al., 2002b). To examine Mtw1p more closely, we tagged and purified both Mtw1p and Nnf1p. The silver-stained gel (Fig. 1 A) of these purifications shows that an identical set of polypeptides, dominated by four prominent bands corresponding to Mtw1p, Nnf1p, Dsn1p, and Nsl1p, copurified in both cases. Densitometric analysis of a Coomassie-stained gel indicated that these proteins are present in an equimolar ratio, suggesting that they form a distinct kinetochore subcomplex. Therefore, we will refer to this group of proteins as the Mtw1 complex. In addition to these four proteins, mass spectrometric analysis of the Mtw1p and Nnf1p purifications revealed the presence of substoichiometric amounts of Mif2p, confirming its association with the Mtw1 complex. Although small amounts of histones were recovered, the full complement of proteins comprising the Cse4p nucleosome was absent from either sample, suggesting that the Mtw1 complex is more distal from centromeric DNA than Mif2p. Interestingly, mass spectrometric analysis also detected the presence of 9 (Mcm22p, Ame1p, Nkp1p, Ctf19p, Okp1p, Mcm21p, Ctf3p, Chl4p, and Iml3p) of the 11 subunits of the central kinetochore Ctf19 complex that we described previously (Cheeseman et al., 2002b). In addition, we detected the Spc25p subunit of the Ndc80 complex (Janke et al., 2001; Wigge and Kilmartin, 2001) as copurifying with Mtw1p, suggesting a physical association between the Mtw1 complex and a subunit of the central kinetochore Ndc80 complex.
Strikingly, comparing the overlapping sets of copurifying proteins defines a path of connectivity from centromeric DNA to the protein complexes of the central and outer kinetochore (Fig. 1 C). For example, whereas the Mif2p sample copurified with the centromeric nucleosome and the Mtw1 complex, purification of the Mtw1 complex resulted in coisolation of Mif2p and components of the Ctf19 and Ndc80 complexes, but not of the complete centromeric nucleosome, indicating that we had moved one step away from centromeric DNA. Similarly, our previous purification of the Ctf19 complex yielded a small amount of Mtw1p and Nnf1p (Cheeseman et al., 2002b; unpublished data).
Nnf1 displays homology to metazoan CENP-H
Recently a human homologue of Mtw1p, hMis12, was shown to be essential for faithful chromosome segregation (Goshima et al., 2003). Using the Block Maker (Henikoff et al., 1995) and motif alignment and search tools (MASTs), we identified significant homology between the Mtw1 complex subunit Nnf1p and CENP-H (Fig. 2). CENP-H is a constitutive component of the metazoan inner kinetochore that colocalizes with CENP-A and -C (Sugata et al., 2000; Fukagawa et al., 2001). The homology between the Saccharomyces cerevisiae Nnf1p and human CENP-H protein sequences (17.4% identity and 49% homology over the entire protein) is similar to the level of homology observed between Mtw1p and hMis12 (19% identity and 49% homology). In addition, the overall P value from the MAST was 2.4 x 10-7, similar to the P value of 4.3 x 10-6 obtained from the Mtw1/hMis12 search. We also note that all of the fungal Nnf1 proteins, as well as the vertebrate CENP-H members, are roughly the same size (200 aa) and feature a central coiled-coil domain (residues 60100 and 120175 in scNnf1, residues 50200 in hsCENP-H). Thus, at least two subunits of the Mtw1 complex have been conserved throughout evolution.
|
|
|
|
We conclude that phosphorylation plays a critical role in Mif2 function. Mif2p is likely regulated by both Ipl1p and by at least one other, as yet unidentified, kinase because serine 325 lies within an acidic sequence, which is not characteristic of an Ipl1p site. The analysis presented here brings the total number of in vivo Ipl1p targets at the kinetochore to eight (Ipl1p, Dam1p, Spc34p, Ask1p, Ndc80p, Sli15p, Mif2p, and Dsn1p). Interestingly, these targets are distributed throughout the inner, central, and outer kinetochore, suggesting that Ipl1p acts at multiple levels to regulate kinetochore structure and function. Intriguingly, based on the phenotypes observed for the mif2 phosphorylation site mutants, Mif2p phosphorylation by Ipl1p appears to play a distinct role from the phosphorylation of the Dam1 complex (Cheeseman et al., 2002b). This suggests that phosphorylation of Mif2p may not be part of the Dam1p pathway required for the establishment of chromosome biorientation (Cheeseman et al., 2002b), but instead contributes to a distinct aspect of kinetochore regulation.
An updated model for the budding yeast kinetochore
Fig. 5 represents an updated model of the budding yeast kinetochore incorporating the information presented in this paper. Interestingly, whereas the CBF3 complex is essential for kinetochore function in budding yeast, our purifications did not reveal significant physical interactions between CBF3 and other kinetochore proteins. No obvious homologues of the CBF3 complex have been detected in Schizosaccharomyces pombe or in metazoans, suggesting that its role may be specialized for the short, nucleotide sequence-constrained centromere unique to budding yeast. In contrast, our purifications show that the interactions establishing connectivity between centromeric DNA and central/outer kinetochore complexes are mediated by conserved proteins consisting of Cse4p/CENP-A, Mif2p/CENP-C, Mtw1p/hMis12, Nnf1p/CENP-H, Nuf2p/hNuf2, and Ndc80/HEC. Fig. 5 B highlights this conserved protein core. It is tempting to speculate that the much larger vertebrate kinetochore is a repetitive expansion of this proposed single core present in budding yeast.
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Complex purifications
Purification of kinetochore complexes was conducted as described previously (Cheeseman et al., 2001) except that 300 mM KCl was used throughout the study. Identification of proteins and phosphorylation sites by mass spectrometry were performed as described previously (Cheeseman et al., 2001; MacCoss et al., 2002). In vitro phosphorylation of kinetochore complexes by GST-Ipl1p from E. coli was performed as described previously (Cheeseman et al., 2002b).
Sequence characterization
Fungal homologues of Nnf1p were identified using basic local alignment search tool searches of the Candida albicans (http://www-sequence.stanford.edu/group/candida), S. pombe (http://www.sanger.ac.uk/Projects/S_pombe), Neurospora crassa (http://www-genome.wi.mit.edu/cgi-bin/annotation/neurospora/blast_page.cgi?organismName=Neurospora), and Magnaporthe grisea (http://www-genome.wi.mit.edu/cgi-bin/annotation/magnaporthe/blast_page.cgi?organismName=Magnaporthe) web sites. Block Maker (http://www.blocks.fhcrc.org/blockmkr/make_blocks.html) was used to align conserved blocks of the fungal sequences. MAST (http://meme.sdsc.edu/meme/website/mast-intro.html) was used to search the nonredundant database for high-scoring sequences with two generated blocks as input. Coiled coils were identified using the COILS server (http://www.ch.embnet.org/software/COILS_form.html). All programs were used with default settings.
ChIP
Immunoprecipitation of formaldehyde cross-linked chromatin was performed as described previously (Enquist-Newman et al., 2001) with the following modifications: immunoprecipitations on TAP-tagged strains were conducted overnight with 0.1 mg/ml rabbit Ig G (Sigma-Aldrich) at 4°C. Immune complexes were subsequently isolated on protein ASepharose CL-4B beads (Amersham Biosciences). PCR used BIO-X-ACT polymerase (Bioline) and were typically run for 29 cycles. PCR products were resolved on 2.5% agarose gels and visualized with ethidium bromide. Stained gels were quantified using a Gel-Doc 1000 system (Bio-Rad Laboratories) and ImageQuant software (Molecular Dynamics).
Immunofluorescence microscopy
Indirect immunofluorescence microscopy on intact yeast cells was performed as described previously (Cheeseman et al., 2002b). The YOL134 antitubulin antibody (Accurate Chemical and Scientific Corporation) was used at a dilution of 1:200. Fluorescein-conjugated anti-IgG heavy chain secondary antibodies (Cappel/Organon Technika Inc. or Jackson Laboratory) were used at 1:500. Light microscopy was performed using a microscope (model TE300; Nikon) equipped with a 100x/1.4 Plan-Apo objective and a cooled CCD camera (model Orca-100; Hamamatsu) controlled by Phase-3 software (Phase-3 Imaging Systems).
Online supplemental material
Fig. S1 shows a Western analysis of purified Mif2p complex with a histone H3-specific antibody (provided by P. Kaufman and J. Sharp, University of California, Berkeley, CA). Fig. S2 provides a ChIP analysis of the proteins Nnf1p, Nsl1p, and Dsn1p. Fig. S3 confirms the presence of TAP-tagged proteins in various mutant backgrounds by Western blotting. Table S1 provides a list of the yeast strains used in this paper. Online supplemental material is available at available at http://www.jcb.org/cgi/content/full/10.1083/jcb.200305100/DC1.
![]() |
Acknowledgments |
---|
This work was supported by grants from the National Institute of General Medical Sciences to G. Barnes (GM-47842); a grant to T. Davis and the Yeast Research Center from the National Center for Research Resources of the National Institutes of Health (Comprehensive Biology: Exploiting the Yeast Genome; PHS P41 RR11823); a National Science Foundation Graduate Research Fellowship to I.M. Cheeseman; and a fellowship of the Deutsche Forschungsgemeinschaft to S. Westermann.
Submitted: 21 May 2003
Accepted: 9 September 2003
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cheeseman, I.M., C. Brew, M. Wolyniak, A. Desai, S. Anderson, N. Muster, J.R. Yates, T.C. Huffaker, D.G. Drubin, and G. Barnes. 2001. Implication of a novel multiprotein Dam1p complex in outer kinetochore function. J. Cell Biol. 155:11371146.
Cheeseman, I.M., D.G. Drubin, and G. Barnes. 2002a. Simple centromere, complex kinetochore: linking spindle microtubules and centromeric DNA in budding yeast. J. Cell Biol. 157:199203.
Cheeseman, I.M., S. Anderson, M. Jwa, E.M. Green, J. Kang, J.R. Yates, C.S.M. Chan, D.G. Drubin, and G. Barnes. 2002b. Phospho-regulation of kinetochore-microtubule attachments by the Aurora kinase Ipl1p. Cell. 111:163172.[Medline]
Cleveland, D.W., Y. Mao, and K.F. Sullivan. 2003. Centromeres and kinetochores: from epigenetics to mitotic checkpoint signalling. Cell. 112:407421.[Medline]
Enquist-Newman, M., I.M. Cheeseman, D. Van Goor, D.G. Drubin, P. Meluh, and G. Barnes. 2001. Dad1p, third component of the Duo1p/Dam1p complex involved in kinetochore function and mitotic spindle integrity. Mol. Biol. Cell. 12:26012613.
Euskirchen, G.M. 2002. Nnf1p, Dsn1p, Mtw1p and Nsl1p: a new group of proteins important for chromosome segregation in Saccharomyces cerevisiae. Eukaryot. Cell. 1:229240.
Fukagawa, T., Y. Mikami, A. Nishihashi, V. Regnier, T. Haraguchi, Y. Hiraoka, N. Sugata, K. Todokoro, W. Brown, and T. Ikemura. 2001. CENP-H, a constitutive centromere component, is required for centromere targeting of CENP-C in vertebrate cells. EMBO J. 20:46034617.
Goshima, G., and M. Yanagida. 2000. Establishing biorientation occurs with precocious separation of the sister kinetochores, but not the arms, in the early spindle of budding yeast. Cell. 100:619633.[Medline]
Goshima, G., T. Kiyomitsu, K. Yoda, and M. Yanagida. 2003. Human centromere chromatin protein hMis12, essential for equal segregation, is independent of CENP-A loading pathway. J. Cell Biol. 160:2539.
He, X., D.R. Rines, C.W. Espelin, and P.K. Sorger. 2001. Molecular analysis of kinetochore-microtubule attachment in budding yeast. Cell. 106:195206.[Medline]
Henikoff, S., J.G. Henikoff, W.J. Alford, and S. Pietrokovski. 1995. Automated construction and graphical presentation of protein blocks from unaligned sequences. Gene. 163:GC17GC26.[CrossRef][Medline]
Howman, E.V., K.J. Fowler, A.J. Newson, S. Redward, A.C. MacDonald, P. Kalitsis, and K.H. Choo. 2000. Early disruption of centromeric chromatin organization in centromere protein A (CENP-A) null mice. Proc. Natl. Acad. Sci. USA. 97:11481153.
Janke, C., J. Ortiz, J. Lechner, A. Shevchenko, M.M. Magiera, C. Schramm, and E. Schiebel. 2001. The budding yeast proteins Spc24p and Spc25p interact with Ndc80p and Nuf2p at the kinetochore and are important for kinetochore clustering and checkpoint control. EMBO J. 20:777791.
Janke, C., J. Ortiz, T.U. Tanaka, J. Lechner, and E. Schiebel. 2002. Four new subunits of the Dam1-Duo1 complex reveal novel functions in sister kinetochore biorientation. EMBO J. 21:181193.
Lechner, J., and J. Carbon. 1991. A 240 kd multisubunit protein complex, CBF3, is a major component of the budding yeast centromere. Cell. 64:717725.[Medline]
Li, Y., J. Bachant, A.A. Alcasabas, Y. Wang, J. Qin, and S.J. Elledge. 2002. The mitotic spindle is required for loading of the DASH complex onto the kinetochore. Genes Dev. 16:183197.
MacCoss, M.J., W.H. McDonald, A. Saraf, R. Sadygov, J.M. Clark, J.J. Tasto, K.L. Gould, D. Wolters, M. Washburn, A. Weiss, et al. 2002. Shotgun identification of potein modifications from protein complexes and lens tissue. Proc. Natl. Acad. Sci. USA. 99:79007905.
Measday, V., D.W. Hailey, I. Pot, S. Givan, K.M. Hyland, G. Cagney, S. Fields, T.N. Davis, and P. Hieter. 2002. Ctf3p, the Mis6 budding yeast homolog, interacts with Mcm22p and Mcm16p at the yeast outer kinetochore. Genes Dev. 16:101113.
Mellor, J., W. Jiang, M. Funk, J. Rathjen, C.A. Barnes, T. Hinz, J.H. Hegemann, and P. Philippsen. 1990. CPF1, a yeast protein which functions in centromeres and promoters. EMBO J. 9:40174026.[Abstract]
Meluh, P.B., and D. Koshland. 1995. Evidence that the MIF2 gene of Saccharomyces cerevisiae encodes a centromere protein with homology to the mammalian centromere protein CENP-C. Mol. Biol. Cell. 6:793807.[Abstract]
Meluh, P.B., and D. Koshland. 1997. Budding yeast centromere composition and assembly as revealed by in vivo cross-linking. Genes Dev. 11:34013412.
Meluh, P.B., P. Yang, L. Glowczewski, D. Koshland, and M.M. Smith. 1998. Cse4p is a component of the core centromere of Saccharomyces cerevisiae. Cell. 94:607613.[Medline]
Ortiz, J., O. Stemmann, S. Rank, and J. Lechner. 1999. A putative protein complex consisting of Ctf19, Mcm21, and Okp1 represents a missing link in the budding yeast kinetochore. Genes Dev. 13:11401155.
Pot, I., V. Measday, B. Snydsman, G. Cagney, S. Fields, T.N. Davis, E.G.D. Miller, and P. Hieter. 2003. Chl4p and Iml3p are two new members of the budding yeast outer kinetochore. Mol. Biol. Cell. 14:460476.
Russell, I.D., A.S. Grancell, and P.K. Sorger. 1999. The unstable F-Box protein p58-Ctf13 forms the structural core of the CBF3 kinetochore complex. J. Cell Biol. 145:933950.
Shang, C., T.Z. Hazburn, I.M. Cheeseman, J. Aranda, S. Fields, D.G. Drubin, and G. Barnes. 2003. Kinetochore protein interactions and their regulation by the Aurora kinase Ipl1p. Mol. Biol. Cell. 14:33423355.
Sharp, J.A., A.A. Franco, M.A. Osley, and P.D. Kaufman. 2002. Chromatin assembly factor I and Hir proteins contribute to building functional kinetochores in S. cerevisiae. Genes Dev. 16:85100.
Stoler, S., K.C. Keith, K.E. Curnick, and M. Fitzgerald-Hayes. 1995. A mutation in CSE4, an essential gene encoding a novel chromatin-associated protein in yeast, causes chromosome nondisjunction and cell cycle arrest at mitosis. Genes Dev. 9:573586.[Abstract]
Sugata, N., S. Li, W.C. Earnshaw, T.J. Yen, K. Yoda, H. Masumoto, E. Munekata, P.E. Warburton, and K. Todokoro. 2000. Human CENP-H multimers colocalize with CENP-A and CENP-C at active centromere-kinetochore complexes. Hum. Mol. Genet. 9:29192926.
Van Hooser, A.A., I.I. Ouspenski, H.C. Gregson, D.A. Starr, T.J. Yen, M.L. Goldberg, K. Yokomori, W.C. Earnshaw, K.F. Sullivan, and B.R. Brinkley. 2001. Specification of kinetochore-forming chromatin by the histone H3 variant CENP-A. J. Cell Sci. 114:35293542.
Wigge, P.A., and J.V. Kilmartin. 2001. The Ndc80p complex from Saccharomyces cerevisiae contains conserved centromere components and has a function in chromosome segregation. J. Cell Biol. 152:349360.