RESUMEN
Mdn1 is an essential AAA (ATPase associated with various activities) protein that removes assembly factors from distinct precursors of the ribosomal 60S subunit. However, Mdn1's large size (â¼5,000 amino acid [aa]) and its limited homology to other well-studied proteins have restricted our understanding of its remodeling function. Here, we present structures for S. pombe Mdn1 in the presence of AMPPNP at up to â¼4 Å or ATP plus Rbin-1, a chemical inhibitor, at â¼8 Å resolution. These data reveal that Mdn1's MIDAS domain is tethered to its ring-shaped AAA domain through an â¼20 nm long structured linker and a flexible â¼500 aa Asp/Glu-rich motif. We find that the MIDAS domain, which also binds other ribosome-assembly factors, docks onto the AAA ring in a nucleotide state-specific manner. Together, our findings reveal how conformational changes in the AAA ring can be directly transmitted to the MIDAS domain and thereby drive the targeted release of assembly factors from ribosomal 60S-subunit precursors.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/química , Simulación de Dinámica Molecular , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/enzimología , Secuencias de Aminoácidos , Animales , Sitios de Unión , Microscopía por Crioelectrón , Biogénesis de Organelos , Unión Proteica , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Células Sf9 , SpodopteraRESUMEN
All cellular proteins are synthesized by ribosomes, whose biogenesis in eukaryotes is a complex multi-step process completed within minutes. Several chemical inhibitors of ribosome function are available and used as tools or drugs. By contrast, we lack potent validated chemical probes to analyze the dynamics of eukaryotic ribosome assembly. Here, we combine chemical and genetic approaches to discover ribozinoindoles (or Rbins), potent and reversible triazinoindole-based inhibitors of eukaryotic ribosome biogenesis. Analyses of Rbin sensitivity and resistance conferring mutations in fission yeast, along with biochemical assays with recombinant proteins, provide evidence that Rbins' physiological target is Midasin, an essential â¼540-kDa AAA+ (ATPases associated with diverse cellular activities) protein. Using Rbins to acutely inhibit or activate Midasin function, in parallel experiments with inhibitor-sensitive or inhibitor-resistant cells, we uncover Midasin's role in assembling Nsa1 particles, nucleolar precursors of the 60S subunit. Together, our findings demonstrate that Rbins are powerful probes for eukaryotic ribosome assembly.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Subunidades Ribosómicas Grandes de Eucariotas/efectos de los fármacos , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Proteínas de Schizosaccharomyces pombe/antagonistas & inhibidores , Triazinas/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Indoles/química , Indoles/aislamiento & purificación , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/metabolismo , Relación Estructura-Actividad , Triazinas/química , Triazinas/aislamiento & purificaciónRESUMEN
Chemical modifications of histones, such as lysine acetylation and ubiquitination, play pivotal roles in epigenetic regulation of gene expression. Methods to alter the epigenome thus hold promise as tools for elucidating epigenetic mechanisms and as therapeutics. However, an entirely chemical method to introduce histone modifications in living cells without genetic manipulation is unprecedented. Here, we developed a chemical catalyst, PEG-LANA-DSSMe 11, that binds with nucleosome's acidic patch and promotes regioselective, synthetic histone acetylation at H2BK120 in living cells. The size of polyethylene glycol in the catalyst was a critical determinant for its in-cell metabolic stability, binding affinity to histones, and high activity. The synthetic acetylation promoted by 11 without genetic manipulation competed with and suppressed physiological H2B ubiquitination, a mark regulating chromatin functions, such as transcription and DNA damage response. Thus, the chemical catalyst will be a useful tool to manipulate epigenome for unraveling epigenetic mechanisms in living cells.
Asunto(s)
Epigenoma , Glicoconjugados/química , Histonas/química , Lisina/química , Polietilenglicoles/química , Procesamiento Proteico-Postraduccional , Acetilación , Catálisis , Ingeniería Química/métodos , Epigénesis Genética , Células HeLa , Histonas/metabolismo , Humanos , Lisina/metabolismo , Nucleosomas/química , Nucleosomas/metabolismo , Oligopéptidos/química , UbiquitinaciónRESUMEN
Pathogenic protein aggregates, called amyloids, are etiologically relevant to various diseases, including neurodegenerative Alzheimer disease. Catalytic photooxygenation of amyloids, such as amyloid-ß (Aß), reduces their toxicity; however, the requirement for light irradiation may limit its utility in large animals, including humans, due to the low tissue permeability of light. Here, we report that Cypridina luciferin analogs, dmCLA-Cl and dmCLA-Br, promoted selective oxygenation of amyloids through chemiexcitation without external light irradiation. Further structural optimization of dmCLA-Cl led to the identification of a derivative with a polar carboxylate functional group and low cellular toxicity: dmCLA-Cl-acid. dmCLA-Cl-acid promoted oxygenation of Aß amyloid and reduced its cellular toxicity without photoirradiation. The chemiexcited oxygenation developed in this study may be an effective approach to neutralizing the toxicity of amyloids, which can accumulate deep inside the body, and treating amyloidosis.
Asunto(s)
Oxígeno , Humanos , Oxígeno/química , Oxígeno/metabolismo , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/química , Amiloide/metabolismo , Amiloide/química , Estructura Molecular , AnimalesRESUMEN
Selective methods for introducing protein post-translational modifications (PTMs) within living cells have proven valuable for interrogating their biological function. In contrast to enzymatic methods, abiotic catalysis should offer access to diverse and new-to-nature PTMs. Herein, we report the boronate-assisted hydroxamic acid (BAHA) catalyst system, which comprises a protein ligand, a hydroxamic acid Lewis base, and a diol moiety. In concert with a boronic acid-bearing acyl donor, our catalyst leverages a local molarity effect to promote acyl transfer to a target lysine residue. Our catalyst system employs micromolar reagent concentrations and affords minimal off-target protein reactivity. Critically, BAHA is resistant to glutathione, a metabolite which has hampered many efforts toward abiotic chemistry within living cells. To showcase this methodology, we installed a variety of acyl groups in E. coli dihydrofolate reductase expressed within human cells. Our results further establish the well-known boronic acid-diol complexation as a bona fide bio-orthogonal reaction with applications in chemical biology and in-cell catalysis.
Asunto(s)
Compuestos de Boro/farmacología , Ácidos Hidroxámicos/química , Tetrahidrofolato Deshidrogenasa/metabolismo , Compuestos de Boro/química , Catálisis , Línea Celular , Escherichia coli/enzimología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
Here, we screened a 10,371 library of diverse molecules using a drug-sensitive fission yeast strain to identify compounds which cause defects in chromosome segregation during mitosis. We identified a phosphorium-ylide-based compound Cutin-1 which inhibits nuclear envelope expansion and nuclear elongation during the closed mitosis of fission yeast, and showed that its target is the ß-subunit of fatty acid synthase. A point mutation in the dehydratase domain of Fas1 conferred in vivo and in vitro resistance to Cutin-1. Time-lapse photomicrography showed that the bulk of the chromosomes were only transiently separated during mitosis, and nucleoli separation was defective. Subsequently sister chromatids re-associated leading to chromosomal mis-segregation. These segregation defects were reduced when the nuclear volume was increased and were increased when the nuclear volume was reduced. We propose that there needs to be sufficient nuclear volume to allow the nuclear elongation necessary during a closed mitosis to take place for proper chromosome segregation, and that inhibition of fatty acid synthase compromises nuclear elongation and leads to defects in chromosomal segregation.
Asunto(s)
Cromosomas Fúngicos/genética , Mitosis , Membrana Nuclear/metabolismo , Schizosaccharomyces/citología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Segregación Cromosómica , Cromosomas Fúngicos/metabolismo , Membrana Nuclear/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
Lysine acylation of proteins is a crucial chemical reaction, both as a post-translational modification and as a method for bioconjugation. We previously developed a chemical catalyst, DSH, which activates a chemically stable thioester including acyl-CoA, allowing the site-selective lysine acylation of histones under physiological conditions. However, a more active catalyst is required for efficient lysine acylation in more complex biological milieu, such as in living cells, but there are no rational guidelines for developing efficient lysine acylation catalysts for use under physiological conditions as opposed to in organic solvents. We, herein, conducted a kinetic analysis of the ability of DSH and several derivatives to mediate lysine acetylation to better understand the structural elements essential for high acetylation activity under physiological conditions. Interestingly, the obtained trend in reactivity was different from that observed in organic solvents, suggesting that a different principle is necessary for designing chemical catalysts specifically for use under physiological conditions compared to catalysts for use in organic solvents. Based on the obtained information, we identified a new catalyst scaffold with high activity and structural flexibility for further modification to improve this catalyst system.
Asunto(s)
Lisina/química , Acetilación , Catálisis , Histonas/metabolismo , Cinética , Relación Estructura-ActividadRESUMEN
Posttranslational modifications (PTMs) of histones play an important role in the complex regulatory mechanisms governing gene transcription, and their dysregulation can cause diseases such as cancer. The lack of methods for site-selectively modifying native chromatin, however, limits our understanding of the functional roles of a specific histone PTM, not as a single mark, but in the intertwined PTM network. Here, we report a synthetic catalyst DMAP-SH (DSH), which activates chemically stable thioesters (including acetyl-CoA) under physiological conditions and transfers various acyl groups to the proximate amino groups. Our data suggest that DSH, conjugated with a nucleosome ligand, such as pyrrole-imidazole-polyamide and LANA (latency-associated nuclear antigen)-peptide, promotes both natural (including acetylation, butyrylation, malonylation, and ubiquitination) and non-natural (azido- and phosphoryl labeling) PTMs on histones in recombinant nucleosomes and/or in native chromatin, at lysine residues close to the DSH moiety. To investigate the validity of our method, we used LANA-DSH to promote histone H2B lysine-120 (K120) acylation, the function of which is largely unknown. H2BK120 acetylation and malonylation modulated higher-order chromatin structures by reducing internucleosomal interactions, and this modulation was further enhanced by histone tail acetylation. This approach, therefore, may have versatile applications for dissecting the regulatory mechanisms underlying chromatin function.
Asunto(s)
Cromatina/química , Histonas/química , Procesamiento Proteico-Postraduccional , Acetilación , Catálisis , Modelos Moleculares , EstereoisomerismoRESUMEN
Post-translational modification of histone tails plays critical roles in gene regulation. Thus, molecules recognizing histone tails and controlling their epigenetic modification are desirable as biochemical tools to elucidate regulatory mechanisms. There are, however, only a few synthetic ligands that bind to histone tails with substantial affinity. We report CA2 and CA3, which exhibited sub-micromolar affinity to histone tails (especially tails with a trimethylated lysine). Multivalent display of trisulfonated calix[4]arene was important for strong binding. CA2 was applicable not only to synthetic tail peptides but also to endogenous histone proteins, and was successfully used to pull-down endogenous histones from nuclear extract. These findings indicate the utility of these supramolecular ligands as biochemical tools for studying chromatin regulator protein and as a targeting motif in ligand-directed catalysis to control epigenetic modifications.
Asunto(s)
Calixarenos/química , Histonas/química , Fenoles/química , Secuencia de Aminoácidos , Biotina/química , Calixarenos/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Cinética , Ligandos , Datos de Secuencia Molecular , Fenoles/metabolismo , Unión Proteica , Resonancia por Plasmón de SuperficieRESUMEN
Indolotryptoline natural products represent a small family of structurally unique chromopyrrolic acid-derived antiproliferative agents. Like many prospective anticancer agents before them, the exploration of their potential clinical utility has been hindered by the limited information known about their mechanism of action. To study the mode of action of two closely related indolotryptolines (BE-54017, cladoniamide A), we selected for drug resistant mutants using a multidrug resistance-suppressed (MDR-sup) Schizosaccharomyces pombe strain. As fission yeast maintains many of the basic cancer-relevant cellular processes present in human cells, it represents an appealing model to use in determining the potential molecular target of antiproliferative natural products through resistant mutant screening. Full genome sequencing of resistant mutants identified mutations in the c and c' subunits of the proteolipid substructure of the vacuolar H(+)-ATPase complex (V-ATPase). This collection of resistance-conferring mutations maps to a site that is distant from the nucleotide-binding sites of V-ATPase and distinct from sites found to confer resistance to known V-ATPase inhibitors. Acid vacuole staining, cross-resistance studies, and direct c/c' subunit mutagenesis all suggest that indolotryptolines are likely a structurally novel class of V-ATPase inhibitors. This work demonstrates the general utility of resistant mutant selection using MDR-sup S. pombe as a rapid and potentially systematic approach for studying the modes of action of cytotoxic natural products.
Asunto(s)
Productos Biológicos/farmacología , Resistencia a Múltiples Medicamentos/genética , Alcaloides Indólicos/farmacología , Mutación/genética , Proteolípidos/genética , ATPasas de Translocación de Protón Vacuolares/genética , Carbolinas/farmacología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Indoles/farmacología , Subunidades de Proteína/química , Subunidades de Proteína/genética , Proteolípidos/química , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/enzimología , ATPasas de Translocación de Protón Vacuolares/químicaRESUMEN
Aberrant aggregates of amyloid-ß (Aß) and tau protein (tau), called amyloid, are related to the etiology of Alzheimer disease (AD). Reducing amyloid levels in AD patients is a potentially effective approach to the treatment of AD. The selective degradation of amyloids via small molecule-catalyzed photooxygenation in vivo is a leading approach; however, moderate catalyst activity and the side effects of scalp injury are problematic in prior studies using AD model mice. Here, leuco ethyl violet (LEV) is identified as a highly active, amyloid-selective, and blood-brain barrier (BBB)-permeable photooxygenation catalyst that circumvents all of these problems. LEV is a redox-sensitive, self-activating prodrug catalyst; self-oxidation of LEV through a hydrogen atom transfer process under photoirradiation produces catalytically active ethyl violet (EV) in the presence of amyloid. LEV effectively oxygenates human Aß and tau, suggesting the feasibility for applications in humans. Furthermore, a concept of using a hydrogen atom as a caging group of a reactive catalyst functional in vivo is postulated. The minimal size of the hydrogen caging group is especially useful for catalyst delivery to the brain through BBB.
Asunto(s)
Enfermedad de Alzheimer , Profármacos , Animales , Profármacos/farmacología , Ratones , Enfermedad de Alzheimer/metabolismo , Catálisis , Modelos Animales de Enfermedad , Péptidos beta-Amiloides/metabolismo , Barrera Hematoencefálica/metabolismo , Humanos , Proteínas tau/metabolismo , Proteínas tau/químicaRESUMEN
Posttranslational modifications (PTMs) of histones, such as lysine acetylation and ubiquitination, regulate chromatin structure and gene expression. In living organisms, histone PTMs are catalyzed by histone-modifying enzymes. Here, we describe an entirely chemical method to introduce histone modifications in living cells without genetic manipulation. The chemical catalyst PEG-LANA-DSSMe activates a thioester acetyl donor, N,S-diacetylcysteamine (NAC-Ac), and promotes regioselective, synthetic histone acetylation at H2BK120 in living cells.
Asunto(s)
Histonas , Procesamiento Proteico-Postraduccional , Acetilación , Catálisis , Histonas/metabolismo , Lisina/metabolismoRESUMEN
Catalytic photo-oxygenation of tau amyloid is a potential therapeutic approach to tauopathies, including Alzheimer disease (AD). However, tau is a complex target containing great molecular size and heterogeneous isoforms/proteoforms. Although catalytic photo-oxygenation has been confirmed when using catalyst 1 and recombinant tau pretreated with heparin, its effects on tau from human patients have not yet been clarified. In this study, focusing on the histidine residues being oxygenated, we have constructed two assay systems capable of quantitatively evaluating the catalytic activity when used on human patient tau: (1) fluorescence labeling at oxygenated histidine sites and (2) LC-MS/MS analysis of histidine-containing fragments. Using these assays, we identified 2 as a promising catalyst for oxygenation of human tau. In addition, our results suggest that aggregated tau induced by heparin is different from actual AD patient tau in developing effective photo-oxygenation catalysts.
Asunto(s)
Enfermedad de Alzheimer , Tauopatías , Humanos , Enfermedad de Alzheimer/metabolismo , Proteínas tau/metabolismo , Cromatografía Liquida , Histidina , Espectrometría de Masas en Tándem , Tauopatías/metabolismoRESUMEN
In the cytoplasm, filamentous actin (F-actin) plays a critical role in cell regulation, including cell migration, stress fiber formation, and cytokinesis. Recent studies have shown that actin filaments that form in the nucleus are associated with diverse functions. Here, using live imaging of an F-actin-specific probe, superfolder GFP-tagged utrophin (UtrCH-sfGFP), we demonstrated the dynamics of nuclear actin in zebrafish (Danio rerio) embryos. In early zebrafish embryos up to around the high stage, UtrCH-sfGFP increasingly accumulated in nuclei during the interphase and reached a peak during the prophase. After nuclear envelope breakdown (NEBD), patches of UtrCH-sfGFP remained in the vicinity of condensing chromosomes during the prometaphase to metaphase. When zygotic transcription was inhibited by injecting α-amanitin, the nuclear accumulation of UtrCH-sfGFP was still observed at the sphere and dome stages, suggesting that zygotic transcription may induce a decrease in nuclear F-actin. The accumulation of F-actin in nuclei may contribute to proper mitotic progression of large cells with rapid cell cycles in zebrafish early embryos, by assisting in NEBD, chromosome congression, and/or spindle assembly.
Asunto(s)
Actinas , Pez Cebra , Animales , Cromosomas/genética , Mitosis , Citoesqueleto de ActinaRESUMEN
Life emerges from a network of biomolecules and chemical reactions catalyzed by enzymes. As enzyme abnormalities are often connected to various diseases, a chemical catalyst promoting physiologically important intracellular reactions in place of malfunctional endogenous enzymes would have great utility in understanding and treating diseases. However, research into such small-molecule chemical enzyme surrogates remains limited, due to difficulties in developing a reactive catalyst capable of activating inert cellular metabolites present at low concentrations. Herein, we report a small-molecule catalyst, mBnA, as a surrogate for a histone acetyltransferase. A hydroxamic acid moiety of suitable electronic characteristics at the catalytic site, paired with a thiol-thioester exchange process, enables mBnA to activate endogenous acyl-CoAs present in low concentrations and promote histone lysine acylations in living cells without the addition of exogenous acyl donors. An enzyme surrogate utilizing cellular metabolites will be a unique tool for elucidation of and synthetic intervention in the chemistry of life and disease.
Asunto(s)
Acilcoenzima A , Histonas , Acilación , Dominio Catalítico , ElectrónicaRESUMEN
Peptides are privileged ligands for diverse biomacromolecules, including proteins; however, their utility is often limited due to low membrane permeability and in-cell instability. Here, we report peptide ligand-inserted eDHFR (PLIED) fusion protein as a universal adaptor for targeting proteins of interest (POI) with cell-permeable and stable synthetic functional small molecules (SFSM). PLIED binds to POI through the peptide moiety, properly orienting its eDHFR moiety, which then recruits trimethoprim (TMP)-conjugated SFSM to POI. Using a lysine-acylating BAHA catalyst as SFSM, we demonstrate that POI (MDM2 and chromatin histone) are post-translationally and synthetically acetylated at specific lysine residues. The residue-selectivity is predictable in an atomic resolution from molecular dynamics simulations of the POI/PLIED/TMP-BAHA (MTX was used as a TMP model) ternary complex. This designer adaptor approach universally enables functional conversion of impermeable peptide ligands to permeable small-molecule ligands, thus expanding the in-cell toolbox of chemical biology.
RESUMEN
Sister chromatid cohesion, mediated by a complex called cohesin, is crucial--particularly at centromeres--for proper chromosome segregation in mitosis and meiosis. In animal mitotic cells, phosphorylation of cohesin promotes its dissociation from chromosomes, but centromeric cohesin is protected by shugoshin until kinetochores are properly captured by the spindle microtubules. However, the mechanism of shugoshin-dependent protection of cohesin is unknown. Here we find a specific subtype of serine/threonine protein phosphatase 2A (PP2A) associating with human shugoshin. PP2A colocalizes with shugoshin at centromeres and is required for centromeric protection. Purified shugoshin complex has an ability to reverse the phosphorylation of cohesin in vitro, suggesting that dephosphorylation of cohesin is the mechanism of protection at centromeres. Meiotic shugoshin of fission yeast also associates with PP2A, with both proteins collaboratively protecting Rec8-containing cohesin at centromeres. Thus, we have revealed a conserved mechanism of centromeric protection of eukaryotic chromosomes in mitosis and meiosis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Emparejamiento Cromosómico , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas de Ciclo Celular/genética , Células HeLa , Humanos , Meiosis , Mitosis , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Fosfoproteínas Fosfatasas/clasificación , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas/metabolismo , Fosforilación , Unión Proteica , Proteína Fosfatasa 2 , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , CohesinasRESUMEN
Fission yeast can be used as a cell-based system for high-throughput drug screening. However, higher drug concentrations are often needed to achieve the same effect as in mammalian cells. Our goal here was to improve drug sensitivity so reduced drugs could be used. Three different methods affecting drug uptakes were tested using an FDA-approved HIV-1 protease inhibitor (PI) drug Darunavir (DRV). First, we tested whether spheroplasts without cell walls increase the drug sensitivity. Second, we examined whether electroporation could be used. Although small improvements were observed, neither of these two methods showed significant increase in the EC50 values of DRV compared with the traditional method. In contrast, when DRV was tested in a mutant strain PR836 that lacks key proteins regulating cellular efflux, a significant increase in the EC50 was observed. A comparison of nine FDA-approved HIV-1 PI drugs between the wild-type RE294 strain and the mutant PR836 strain showed marked enhancement of the drug sensitivities ranging from an increase of 0.56 log to 2.48 logs. Therefore, restricting cellular efflux through the adaption of the described fission yeast mutant strain enhances the drug sensitivity, reduces the amount of drug used, and increases the chance of success in future drug discovery.
RESUMEN
Aurora-B kinases are important regulators of mitotic chromosome segregation, where they are required for the faithful bi-orientation of sister chromatids. In contrast to mitosis, sister chromatids have to be oriented toward the same spindle pole in meiosis-I, while homologous chromosomes are bi-oriented. We find that the fission yeast Aurora kinase Ark1 is required for the faithful bi-orientation of sister chromatids in mitosis and of homologous chromosomes in meiosis-I. Unexpectedly, Ark1 is also necessary for the faithful mono-orientation of sister chromatids in meiosis-I, even though the canonical mono-orientation pathway, which depends on Moa1 and Rec8, seems intact. Our data suggest that Ark1 prevents unified sister kinetochores during metaphase-I from merotelic attachment to both spindle poles and thus from being torn apart during anaphase-I, revealing a novel mechanism promoting monopolar attachment. Furthermore, our results provide an explanation for the previously enigmatic observation that fission yeast Shugoshin Sgo2, which assists in loading Aurora to centromeres, and its regulator Bub1 are required for the mono-orientation of sister chromatids in meiosis-I.
Asunto(s)
Cinetocoros/metabolismo , Meiosis , Proteínas Serina-Treonina Quinasas/fisiología , Schizosaccharomyces/genética , Alelos , Aurora Quinasas , Núcleo Celular/metabolismo , Cromátides/ultraestructura , Proteínas Cromosómicas no Histona/fisiología , Procesamiento de Imagen Asistido por Computador , Mitosis , Modelos Biológicos , Modelos Genéticos , Fosfoproteínas/metabolismo , Schizosaccharomyces/fisiología , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiología , Intercambio de Cromátides HermanasRESUMEN
Meiosis comprises a pair of specialized nuclear divisions that produce haploid germ cells. To accomplish this, sister chromatids must segregate together during the first meiotic division (meiosis I), which requires that sister chromatid cohesion persists at centromeres. The factors that protect centromeric cohesion during meiosis I have remained elusive. Here we identify Sgo1 (shugoshin), a protector of the centromeric cohesin Rec8 in fission yeast. We also identify a homologue of Sgo1 in budding yeast. We provide evidence that shugoshin is widely conserved among eukaryotes. Moreover, we identify Sgo2, a paralogue of shugoshin in fission yeast, which is required for faithful mitotic chromosome segregation. Localization of Sgo1 and Sgo2 at centromeres requires the kinase Bub1, identifying shugoshin as a crucial target for the kinetochore function of Bub1. These findings provide insights into the evolution of meiosis and kinetochore regulation during mitosis and meiosis.