共查询到20条相似文献,搜索用时 15 毫秒
1.
Westermann S Cheeseman IM Anderson S Yates JR Drubin DG Barnes G 《The Journal of cell biology》2003,163(2):215-222
How kinetochore proteins are organized to connect chromosomes to spindle microtubules, and whether any structural and organizational themes are common to kinetochores from distantly related organisms, are key unanswered questions. Here, we used affinity chromatography and mass spectrometry to generate a map of kinetochore protein interactions. The budding yeast CENP-C homologue Mif2p specifically copurified with histones H2A, H2B, and H4, and with the histone H3-like CENP-A homologue Cse4p, strongly suggesting that Cse4p replaces histone H3 in a specialized centromeric nucleosome. A novel four-protein Mtw1 complex, the Nnf1p subunit of which has homology to the vertebrate kinetochore protein CENP-H, also copurified with Mif2p and a variety of central kinetochore proteins. We show that Mif2 is a critical in vivo target of the Aurora kinase Ipl1p. Chromatin immunoprecipitation studies demonstrated the biological relevance of these associations. We propose that a molecular core consisting of CENP-A, -C, -H, and Ndc80/HEC has been conserved from yeast to humans to link centromeres to spindle microtubules. 相似文献
2.
The N terminus of the centromere H3-like protein Cse4p performs an essential function distinct from that of the histone fold domain 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Chen Y Baker RE Keith KC Harris K Stoler S Fitzgerald-Hayes M 《Molecular and cellular biology》2000,20(18):7037-7048
Cse4p is an evolutionarily conserved histone H3-like protein that is thought to replace H3 in a specialized nucleosome at the yeast (Saccharomyces cerevisiae) centromere. All known yeast, worm, fly, and human centromere H3-like proteins have highly conserved C-terminal histone fold domains (HFD) but very different N termini. We have carried out a comprehensive and systematic mutagenesis of the Cse4p N terminus to analyze its function. Surprisingly, only a 33-amino-acid domain within the 130-amino-acid-long N terminus is required for Cse4p N-terminal function. The spacing of the essential N-terminal domain (END) relative to the HFD can be changed significantly without an apparent effect on Cse4p function. The END appears to be important for interactions between Cse4p and known kinetochore components, including the Ctf19p/Mcm21p/Okp1p complex. Genetic and biochemical evidence shows that Cse4p proteins interact with each other in vivo and that nonfunctional cse4 END and HFD mutant proteins can form functional mixed complexes. These results support different roles for the Cse4p N terminus and the HFD in centromere function and are consistent with the proposed Cse4p nucleosome model. The structure-function characteristics of the Cse4p N terminus are relevant to understanding how other H3-like proteins, such as the human homolog CENP-A, function in kinetochore assembly and chromosome segregation. 相似文献
3.
CENP-A exceeds microtubule attachment sites in centromere clusters of both budding and fission yeast
The stoichiometries of kinetochores and their constituent proteins in yeast and vertebrate cells were determined using the histone H3 variant CENP-A, known as Cse4 in budding yeast, as a counting standard. One Cse4-containing nucleosome exists in the centromere (CEN) of each chromosome, so it has been assumed that each anaphase CEN/kinetochore cluster contains 32 Cse4 molecules. We report that anaphase CEN clusters instead contained approximately fourfold more Cse4 in Saccharomyces cerevisiae and ~40-fold more CENP-A (Cnp1) in Schizosaccharomyces pombe than predicted. These results suggest that the number of CENP-A molecules exceeds the number of kinetochore-microtubule (MT) attachment sites on each chromosome and that CENP-A is not the sole determinant of kinetochore assembly sites in either yeast. In addition, we show that fission yeast has enough Dam1-DASH complex for ring formation around attached MTs. The results of this study suggest the need for significant revision of existing CEN/kinetochore architectural models. 相似文献
4.
H Lin P de Carvalho D Kho C Y Tai P Pierre G R Fink D Pellman 《The Journal of cell biology》2001,155(7):1173-1184
The attachment of kinetochores to spindle microtubules (MTs) is essential for maintaining constant ploidy in eukaryotic cells. Here, biochemical and imaging data is presented demonstrating that the budding yeast CLIP-170 orthologue Bik1is a component of the kinetochore-MT binding interface. Strikingly, Bik1 is not required for viability in haploid cells, but becomes essential in polyploids. The ploidy-specific requirement for BIK1 enabled us to characterize BIK1 without eliminating nonhomologous genes, providing a new approach to circumventing the overlapping function that is a common feature of the cytoskeleton. In polyploid cells, Bik1 is required before anaphase to maintain kinetochore separation and therefore contributes to the force that opposes the elastic recoil of attached sister chromatids. The role of Bik1 in kinetochore separation appears to be independent of the role of Bik1 in regulating MT dynamics. The finding that a protein involved in kinetochore-MT attachment is required for the viability of polyploids has potential implications for cancer therapeutics. 相似文献
5.
Cse4 is the budding yeast homologue of CENP-A, a modified histone H3 that specifies the base of kinetochores in all eukaryotes. Budding yeast is unique in having only one kinetochore microtubule attachment site per centromere. The centromere is specified by CEN DNA, a sequence-specific binding complex (CBF3), and a Cse4-containing nucleosome. Here we compare the ratio of kinetochore proximal Cse4-GFP fluorescence at anaphase to several standards including purified EGFP molecules in vitro to generate a calibration curve for the copy number of GFP-fusion proteins. Our results yield a mean of ~5 Cse4s, ~3 inner kinetochore CBF3 complexes, and ~20 outer kinetochore Ndc80 complexes. Our calibrated measurements increase 2.5-3-fold protein copy numbers at eukaryotic kinetochores based on previous ratio measurements assuming two Cse4s per budding yeast kinetochore. All approximately five Cse4s may be associated with the CEN nucleosome, but we show that a mean of three Cse4s could be located within flanking nucleosomes at random sites that differ between chromosomes. 相似文献
6.
CLIP-170 family proteins regulate microtubule plus end dynamics. Two reports published in this issue of Developmental Cell show that Bik1 and tip1p, the CLIP-170-like proteins of budding and fission yeast, are carried to microtubule plus ends by kinesin motor proteins. These findings indicate a complex interplay between microtubule-associated proteins and suggest a novel mechanism by which kinesin proteins stabilize microtubules. 相似文献
7.
During cell division, segregation of sister chromatids to daughter cells is achieved by the poleward pulling force of microtubules, which attach to the chromatids by means of a multiprotein complex, the kinetochore. Kinetochores assemble at the centromeric DNA organized by specialized centromeric nucleosomes. In contrast to other eukaryotes, which typically have large repetitive centromeric regions, budding yeast CEN DNA is defined by a 125 bp sequence and assembles a single centromeric nucleosome. In budding yeast, as well as in other eukaryotes, the Cse4 histone variant (known in vertebrates as CENP-A) is believed to substitute for histone H3 at the centromeric nucleosome. However, the exact composition of the CEN nucleosome remains a subject of debate. We report the use of a novel ChIP approach to reveal the composition of the centromeric nucleosome and its localization on CEN DNA in budding yeast. Surprisingly, we observed a strong interaction of H3, as well as Cse4, H4, H2A, and H2B, but not histone chaperone Scm3 (HJURP in human) with the centromeric DNA. H3 localizes to centromeric DNA at all stages of the cell cycle. Using a sequential ChIP approach, we could demonstrate the co-occupancy of H3 and Cse4 at the CEN DNA. Our results favor a H3-Cse4 heterotypic octamer at the budding yeast centromere. Whether or not our model is correct, any future model will have to account for the stable association of histone H3 with the centromeric DNA. 相似文献
8.
Kinetochores are the specialized protein structures that form on centromeric DNA and direct chromosome segregation. It is critical that all chromosomes assemble a single kinetochore every cell cycle. One hallmark of all eukaryotic kinetochores is CENP-A, an essential centromeric histone H3 (CenH3) variant. Overexpression of CENP-A causes mislocalization to euchromatin, which could lead to deleterious consequences because CENP-A overexpression is associated with colorectal cancer . Although CENP-A protein levels are important for genomic stability, little is known about the mechanisms of CenH3 regulation. Here, we show that the levels of the budding yeast CenH3, Cse4, are regulated by ubiquitin-proteasome-mediated proteolysis. Because mutation of all Cse4 lysine residues did not completely stabilize the protein, we isolated a dominant lethal mutant, CSE4-351, that was stable. The Cse4-351 protein localized to euchromatin, suggesting that proteolysis prevents CenH3 euchromatic localization. When wild-type Cse4 was fused to a degron signal, the soluble Cse4 protein was rapidly degraded, but the centromere bound Cse4 was stable, indicating that centromere localization protects Cse4 from degradation. Taken together, these data identify proteolysis as one mechanism that contributes to the restricted centromere localization of the yeast CenH3. 相似文献
9.
The kinetochore (centromeric DNA and associated proteins) is a key determinant for high fidelity chromosome transmission. Evolutionarily conserved Scm3p is an essential component of centromeric chromatin and is required for assembly and function of kinetochores in humans, fission yeast, and budding yeast. Overexpression of HJURP, the mammalian homolog of budding yeast Scm3p, has been observed in lung and breast cancers and is associated with poor prognosis; however, the physiological relevance of these observations is not well understood. We overexpressed SCM3 and HJURP in Saccharomyces cerevisiae and HJURP in human cells and defined domains within Scm3p that mediate its chromosome loss phenotype. Our results showed that the overexpression of SCM3 (GALSCM3) or HJURP (GALHJURP) caused chromosome loss in a wild-type yeast strain, and overexpression of HJURP led to mitotic defects in human cells. GALSCM3 resulted in reduced viability in kinetochore mutants, premature separation of sister chromatids, and reduction in Cse4p and histone H4 at centromeres. Overexpression of CSE4 or histone H4 suppressed chromosome loss and restored levels of Cse4p at centromeres in GALSCM3 strains. Using mutant alleles of scm3, we identified a domain in the N-terminus of Scm3p that mediates its interaction with CEN DNA and determined that the chromosome loss phenotype of GALSCM3 is due to centromeric association of Scm3p devoid of Cse4p/H4. Furthermore, we determined that similar to other systems the centromeric association of Scm3p is cell cycle regulated. Our results show that altered stoichiometry of Scm3p/HJURP, Cse4p, and histone H4 lead to defects in chromosome segregation. We conclude that stringent regulation of HJURP and SCM3 expression are critical for genome stability. 相似文献
10.
The functional diversity and structural heterogeneity of microtubules are largely determined by microtubule-associated proteins (MAPs) [1] [2]. Bik1p (bilateral karyogamy defect protein) is one of the MAPs required for microtubule assembly, stability and function in cell processes such as karyogamy and nuclear migration and positioning in the yeast Saccharomyces cerevisiae [3]. The macrocyclic immunosuppressive antibiotic rapamycin, complexed with its binding protein FKBP12, binds to and inhibits the target of rapamycin protein (TOR) in yeast [4] [5]. We report here that TOR physically interacts with Bik1p, the yeast homolog of human CLIP-170/Restin [6] [7]. Inhibition of TOR by rapamycin significantly affects microtubule assembly, elongation and stability. This function of TOR is independent of new protein synthesis. Rapamycin also causes defects in spindle orientation, nuclear movement and positioning, karyogamy and chromosomal stability, defects also found in the bikDelta mutant. Our data suggest a role for TOR signaling in regulating microtubule stability and function, possibly through Bik1p. 相似文献
11.
Scm3 is essential to recruit the histone h3 variant cse4 to centromeres and to maintain a functional kinetochore 总被引:3,自引:0,他引:3
The kinetochore is a complex multiprotein structure located at centromeres that is essential for proper chromosome segregation. Budding-yeast Cse4 is an essential evolutionarily conserved histone H3 variant recruited to the centromere by an unknown mechanism. We have identified Scm3, an inner kinetochore protein that immunopurifies with Cse4. Scm3 is essential for viability and localizes to all centromeres. Construction of a conditional SCM3 allele reveals that depletion results in metaphase arrest, with duplicated spindle poles, short spindles, and unequal DNA distribution. The metaphase arrest is mediated by the mitotic spindle checkpoint being dependent on Mad1 and the Aurora kinase B homolog Ipl1. Scm3 interacts with both Ndc10 and Cse4 and is essential to establish centromeric chromatin after DNA replication. In addition, Scm3 is required to maintain kinetochore function throughout the cell cycle. We propose a model in which Ndc10/Scm3 binds to centromeric DNA, which is in turn essential for targeting Cse4 to centromeres. 相似文献
12.
CLIPs are microtubule plus end-associated proteins that mediate interactions required for cell polarity and cell division. Here we demonstrate that budding yeast Bik1, unlike its human ortholog CLIP-170, is targeted to the microtubule plus end by a kinesin-dependent transport mechanism. Bik1 forms a complex with the kinesin Kip2. Fluorescently labeled Bik1 and Kip2 comigrate along individual microtubules. Bik1 exists in distinct intracellular pools: a stable pool at the spindle pole body that is depleted during cell cycle progression, a soluble pool from which Bik1 can be recruited during microtubule initiation, and a dynamic plus end pool maintained by Kip2. Kip2 stabilizes microtubules by targeting Bik1 to the plus end and Kip2 levels are controlled during the cell cycle. As with Bik1, the targeting of dynein to the microtubule plus end requires Kip2. These findings reveal a central role for Kip2-dependent transport in the cell cycle control of microtubule dynamics and dynein-dependent motility. 相似文献
13.
Proper centromere function is critical to maintain genomic stability and to prevent aneuploidy, a hallmark of tumors and birth defects. A conserved feature of all eukaryotic centromeres is an essential histone H3 variant called CENP-A that requires a centromere targeting domain (CATD) for its localization. Although proteolysis prevents CENP-A from mislocalizing to euchromatin, regulatory factors have not been identified. Here, we identify an E3 ubiquitin ligase called Psh1 that leads to the degradation of Cse4, the budding yeast CENP-A homolog. Cse4 overexpression is toxic to psh1Δ cells and results in euchromatic localization. Strikingly, the Cse4 CATD is a key regulator of its stability and helps Psh1 discriminate Cse4 from histone H3. Taken together, we propose that the CATD has a previously unknown role in maintaining the exclusive localization of Cse4 by preventing its mislocalization to euchromatin via Psh1-mediated degradation. 相似文献
14.
15.
Chromosome segregation depends on kinetochore biorientation so that sister kinetochores attach to microtubules from opposite poles and come under tension. The budding yeast Ipl1/Aurora protein kinase allows the absence of tension to activate the spindle checkpoint. We found that checkpoint activation in the mtw1-1 kinetochore mutant requires Ipl1p, suggesting that Mtw1p promotes tension. We isolated mtw1-1 dosage suppressors and identified Dsn1, a kinetochore protein that immunoprecipitates with the Mif2/CENP-C and Cse4/CENP-A proteins, as well as the Mtw1, Nnf1, and Nsl1 kinetochore proteins. mtw1 and dsn1 mutant strains exhibit similar phenotypes, suggesting that Mtw1p and Dsn1p act together. Although mtw1 mutant cells contained unattached chromosomes, attachment was restored by impairing Ipl1p function. These results suggest that mtw1 mutant kinetochores are competent to bind microtubules but Ipl1p generates unattached chromosomes. We therefore propose that an Mtw1 complex is required for kinetochore biorientation that is monitored by the Ipl1p kinase. 相似文献
16.
《Cell cycle (Georgetown, Tex.)》2013,12(19):3369-3378
Centromeres are specialized chromatin domains where kinetochores assemble. Centromeres contain as a conserved feature nucleosomes that are composed of the canonical histones H2A, H2B and H4 and a centromere-specific histone H3 variant, known as CENP-A in humans and Cse4 in budding yeast. The incorporation of CENP-A homologs into centromeric chromatin is cell cycle regulated and is assisted by related assembly factors named Scm3 in yeast and HJURP in human cells. Here, we describe that the budding yeast Scm3 binds weakly to centromeres during interphase including S phase when Cse4 assembles into centromeres. In anaphase Scm3 then becomes 2.5-fold enriched at kinetochores where it is dynamic with a half recovery time t½ of 36 sec. In contrast, Cse4 is stably integrated into kinetochores. In addition, ten Scm3 molecules bind to a cluster of 16 kinetochores with 32 Cse4 molecules suggesting a 1:3 ratio at kinetochores between the two proteins. Analysis of conditional lethal scm3–1 mutant cells indicated that Scm3 participates in maintaining Cse4 at centromeres in anaphase. Thus, Scm3 interacts transiently with kinetochores in anaphase where it safeguards Cse4 even after its S phase incorporation into centromeres. 相似文献
17.
Analysis of Primary Structural Determinants That Distinguish the Centromere-Specific Function of Histone Variant Cse4p from Histone H3
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Kevin C. Keith Richard E. Baker Yinhuai Chen Kendra Harris Sam Stoler Molly Fitzgerald-Hayes 《Molecular and cellular biology》1999,19(9):6130-6139
Cse4p is a variant of histone H3 that has an essential role in chromosome segregation and centromere chromatin structure in budding yeast. Cse4p has a unique 135-amino-acid N terminus and a C-terminal histone-fold domain that is more than 60% identical to histone H3 and the mammalian centromere protein CENP-A. Cse4p and CENP-A have biochemical properties similar to H3 and probably replace H3 in centromere-specific nucleosomes in yeasts and mammals, respectively. In order to identify regions of Cse4p that distinguish it from H3 and confer centromere function, a systematic site-directed mutational analysis was performed. Nested deletions of the Cse4p N terminus showed that this region of the protein contains at least one essential domain. The C-terminal histone-fold domain of Cse4p was analyzed by changing Cse4p amino acids that differ between Cse4p and H3 to the analogous H3 residues. Extensive substitution of contiguous Cse4p residues with H3 counterparts resulted in cell lethality. However, all large lethal substitution alleles could be subdivided into smaller viable alleles, many of which caused elevated rates of mitotic chromosome loss. The results indicate that residues critical for wild-type Cse4p function and high-fidelity chromosome transmission are distributed across the entire histone-fold domain. Our findings are discussed in the context of the known structure of H3 within the nucleosome and compared with previous results reported for CENP-A. 相似文献
18.
During mitosis, sister chromatids congress on both sides of the spindle equator to facilitate the correct partitioning of the genomic material. Chromosome congression requires a finely tuned control of microtubule dynamics by the kinesin motor proteins. In Saccharomyces cerevisiae, the kinesin proteins Cin8, Kip1, and Kip3 have a pivotal role in chromosome congression. It has been hypothesized that additional proteins that modulate microtubule dynamics are involved. Here, we show that the microtubule plus-end tracking protein Bik1—the budding yeast ortholog of CLIP-170—is essential for chromosome congression. We find that nuclear Bik1 localizes to the kinetochores in a cell cycle–dependent manner. Disrupting the nuclear pool of Bik1 with a nuclear export signal (Bik1-NES) leads to slower cell-cycle progression characterized by a delayed metaphase–anaphase transition. Bik1-NES cells have mispositioned kinetochores along the spindle in metaphase. Furthermore, using proximity-dependent methods, we identify Cin8 as an interaction partner of Bik1. Deleting CIN8 reduces the amount of Bik1 at the spindle. In contrast, Cin8 retains its typical bilobed distribution in the Bik1-NES mutant and does not localize to the unclustered kinetochores. We propose that Bik1 functions with Cin8 to regulate kinetochore–microtubule dynamics for correct kinetochore positioning and chromosome congression. 相似文献
19.
Nonhistone Scm3 and histones CenH3-H4 assemble the core of centromere-specific nucleosomes 总被引:4,自引:0,他引:4
The budding yeast histone H3 variant, Cse4, replaces conventional histone H3 in centromeric chromatin and, together with centromere-specific DNA-binding factors, directs assembly of the kinetochore, a multiprotein complex mediating chromosome segregation. We have identified Scm3, a nonhistone protein that colocalizes with Cse4 and is required for its centromeric association. Bacterially expressed Scm3 binds directly to and reconstitutes a stoichiometric complex with Cse4 and histone H4 but not with conventional histone H3 and H4. A conserved acidic domain of Scm3 is responsible for directing the Cse4-specific interaction. Strikingly, binding of Scm3 can replace histones H2A-H2B from preassembled Cse4-containing histone octamers. This incompatibility between Scm3 and histones H2A-H2B is correlated with diminished in vivo occupancy of histone H2B, H2A, and H2AZ at centromeres. Our findings indicate that nonhistone Scm3 serves to assemble and maintain Cse4-H4 at centromeres and may replace histone H2A-H2B dimers in a centromere-specific nucleosome core. 相似文献