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1.
J Biol Chem ; 299(6): 104797, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37156397

RESUMO

Coenzyme Q (CoQ) is an essential component of the electron transport system in aerobic organisms. CoQ10 has ten isoprene units in its quinone structure and is especially valuable as a food supplement. However, the CoQ biosynthetic pathway has not been fully elucidated, including synthesis of the p-hydroxybenzoic acid (PHB) precursor to form a quinone backbone. To identify the novel components of CoQ10 synthesis, we investigated CoQ10 production in 400 Schizosaccharomyces pombe gene-deleted strains in which individual mitochondrial proteins were lost. We found that deletion of coq11 (an S. cerevisiae COQ11 homolog) and a novel gene designated coq12 lowered CoQ levels to ∼4% of that of the WT strain. Addition of PHB or p-hydroxybenzaldehyde restored the CoQ content and growth and lowered hydrogen sulfide production of the Δcoq12 strain, but these compounds did not affect the Δcoq11 strain. The primary structure of Coq12 has a flavin reductase motif coupled with an NAD+ reductase domain. We determined that purified Coq12 protein from S. pombe displayed NAD+ reductase activity when incubated with ethanol-extracted substrate of S. pombe. Because purified Coq12 from Escherichia coli did not exhibit reductase activity under the same conditions, an extra protein is thought to be necessary for its activity. Analysis of Coq12-interacting proteins by LC-MS/MS revealed interactions with other Coq proteins, suggesting formation of a complex. Thus, our analysis indicates that Coq12 is required for PHB synthesis, and it has diverged among species.


Assuntos
NADH NADPH Oxirredutases , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Cromatografia Líquida , NAD/metabolismo , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/isolamento & purificação , NADH NADPH Oxirredutases/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Proteínas de Schizosaccharomyces pombe/metabolismo , Espectrometria de Massas em Tandem , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo
2.
Nucleic Acids Res ; 49(12): 6832-6848, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34157114

RESUMO

Rad51 is the key protein in homologous recombination that plays important roles during DNA replication and repair. Auxiliary factors regulate Rad51 activity to facilitate productive recombination, and prevent inappropriate, untimely or excessive events, which could lead to genome instability. Previous genetic analyses identified a function for Rrp1 (a member of the Rad5/16-like group of SWI2/SNF2 translocases) in modulating Rad51 function, shared with the Rad51 mediator Swi5-Sfr1 and the Srs2 anti-recombinase. Here, we show that Rrp1 overproduction alleviates the toxicity associated with excessive Rad51 levels in a manner dependent on Rrp1 ATPase domain. Purified Rrp1 binds to DNA and has a DNA-dependent ATPase activity. Importantly, Rrp1 directly interacts with Rad51 and removes it from double-stranded DNA, confirming that Rrp1 is a translocase capable of modulating Rad51 function. Rrp1 affects Rad51 binding at centromeres. Additionally, we demonstrate in vivo and in vitro that Rrp1 possesses E3 ubiquitin ligase activity with Rad51 as a substrate, suggesting that Rrp1 regulates Rad51 in a multi-tiered fashion.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Rad51 Recombinase/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adenosina Trifosfatases/metabolismo , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/fisiologia , Instabilidade Genômica , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Proteínas de Schizosaccharomyces pombe/fisiologia
3.
Nat Commun ; 12(1): 3244, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050143

RESUMO

N6-methyladenosine (m6A) is a modification that plays pivotal roles in RNA metabolism and function, although its functions in spliceosomal U6 snRNA remain unknown. To elucidate its role, we conduct a large-scale transcriptome analysis of a Schizosaccharomyces pombe strain lacking this modification and found a global change of pre-mRNA splicing. The most significantly impacted introns are enriched for adenosine at the fourth position pairing the m6A in U6 snRNA, and exon sequences weakly recognized by U5 snRNA. This suggests cooperative recognition of 5' splice site by U6 and U5 snRNPs, and also a role of m6A facilitating efficient recognition of the splice sites weakly interacting with U5 snRNA, indicating that U6 snRNA m6A relaxes the 5' exon constraint and allows protein sequence diversity along with explosively increasing number of introns over the course of eukaryotic evolution.


Assuntos
Regulação Fúngica da Expressão Gênica , Splicing de RNA , RNA Fúngico/metabolismo , RNA Nuclear Pequeno/metabolismo , Schizosaccharomyces/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Éxons/genética , Espectrometria de Massas , Metiltransferases/genética , Metiltransferases/isolamento & purificação , Metiltransferases/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Sítios de Splice de RNA/genética , RNA Fúngico/genética , RNA Nuclear Pequeno/genética , RNA-Seq , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6 , Ribonucleoproteína Nuclear Pequena U5 , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Proteínas de Schizosaccharomyces pombe/metabolismo
4.
Nat Commun ; 10(1): 5764, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848341

RESUMO

The fundamental unit of chromatin, the nucleosome, is an intricate structure that requires histone chaperones for assembly. ATAD2 AAA+ ATPases are a family of histone chaperones that regulate nucleosome density and chromatin dynamics. Here, we demonstrate that the fission yeast ATAD2 homolog, Abo1, deposits histone H3-H4 onto DNA in an ATP-hydrolysis-dependent manner by in vitro reconstitution and single-tethered DNA curtain assays. We present cryo-EM structures of an ATAD2 family ATPase to atomic resolution in three different nucleotide states, revealing unique structural features required for histone loading on DNA, and directly visualize the transitions of Abo1 from an asymmetric spiral (ATP-state) to a symmetric ring (ADP- and apo-states) using high-speed atomic force microscopy (HS-AFM). Furthermore, we find that the acidic pore of ATP-Abo1 binds a peptide substrate which is suggestive of a histone tail. Based on these results, we propose a model whereby Abo1 facilitates H3-H4 loading by utilizing ATP.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/ultraestrutura , Chaperonas de Histonas/ultraestrutura , Nucleossomos/metabolismo , Proteínas de Schizosaccharomyces pombe/ultraestrutura , ATPases Associadas a Diversas Atividades Celulares/isolamento & purificação , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Microscopia Crioeletrônica/métodos , DNA/metabolismo , Chaperonas de Histonas/isolamento & purificação , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Estrutura Quaternária de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Proteínas de Schizosaccharomyces pombe/metabolismo , Imagem Individual de Molécula/métodos
5.
Cell Cycle ; 18(14): 1532-1536, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31219728

RESUMO

The spliceosome is a complex molecular machine assembled from many components, which catalyzes the removal of introns from mRNA precursors. Our previous study revealed that the Nrl1 (NRDE-2 like 1) protein associates with spliceosome proteins and regulates pre-mRNA splicing and homologous recombination-dependent R-loop formation in the fission yeast Schizosaccharomyces pombe. Here, we identify proteins associated with splicing factors Ntr1, Ntr2, Brr2 and Gpl1, a poorly characterized G-patch domain-containing protein required for efficient splicing. This work provides new evidence that Nrl1 and splicing factors physically interact and reveals additional insights into the protein interaction network of the spliceosome. We discuss implications of these findings in the light of recent progress in our understanding of how Nrl1 and splicing factors ensure genome stability.


Assuntos
RNA Helicases/metabolismo , Fatores de Processamento de RNA/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Estruturas R-Loop/genética , RNA Helicases/genética , Splicing de RNA/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/isolamento & purificação , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Spliceossomos/genética , Spliceossomos/metabolismo
6.
Sci Rep ; 9(1): 8618, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197198

RESUMO

Rif1 is a conserved protein regulating replication timing and binds preferentially to the vicinity of late-firing/dormant origins in fission yeast. The Rif1 binding sites on the fission yeast genome have an intrinsic potential to generate G-quadruplex (G4) structures to which purified Rif1 preferentially binds. We previously proposed that Rif1 generates chromatin architecture that may determine replication timing by facilitating the chromatin loop formation. Here, we conducted detailed biochemical analyses on Rif1 and its G4 binding. Rif1 prefers sequences containing long stretches of guanines and binds preferentially to the multimeric G4 of parallel or hybrid/mix topology. Rif1 forms oligomers and binds simultaneously to multiple G4. We present a model on how Rif1 may facilitate the formation of chromatin architecture through its G4 binding and oligomerization properties.


Assuntos
Quadruplex G , Multimerização Proteica , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Sequência de Bases , Cromatina/metabolismo , DNA Fúngico/metabolismo , Modelos Biológicos , Oligonucleotídeos/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Telômero/metabolismo , Proteínas de Ligação a Telômeros/isolamento & purificação
7.
Cell Res ; 27(12): 1503-1520, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29160296

RESUMO

Telomeric shelterin complex caps chromosome ends and plays a crucial role in telomere maintenance and protection. In the fission yeast Schizosaccharomyces pombe, shelterin is composed of telomeric single- and double-stranded DNA-binding protein subcomplexes Pot1-Tpz1 and Taz1-Rap1, which are bridged by their interacting protein Poz1. However, the structure of Poz1 and how Poz1 functions as an interaction hub in the shelterin complex remain unclear. Here we report the crystal structure of Poz1 in complex with Poz1-binding motifs of Tpz1 and Rap1. The crystal structure shows that Poz1 employs two different binding surfaces to interact with Tpz1 and Rap1. Unexpectedly, the structure also reveals that Poz1 adopts a dimeric conformation. Mutational analyses suggest that proper interactions between Tpz1, Poz1, and Rap1 in the shelterin core complex are required for telomere length homeostasis and heterochromatin structure maintenance at telomeres. Structural resemblance between Poz1 and the TRFH domains of other shelterin proteins in fission yeast and humans suggests a model for the evolution of shelterin proteins.


Assuntos
Proteínas de Transporte/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/química , Proteínas de Ligação a Telômeros/química , Telômero/química , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Proteínas de Ligação a DNA , Conformação Proteica , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Complexo Shelterina , Telômero/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/isolamento & purificação
8.
Protein Expr Purif ; 135: 61-69, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28502666

RESUMO

The Ndc80 complex is a conserved outer kinetochore protein complex consisting of Ndc80 (Hec1), Nuf2, Spc24 and Spc25. This complex comprises a major, if not the sole, platform with which the plus ends of the spindle microtubules directly interact. In fission yeast, several studies indicate that multiple microtubule-associated proteins including the Dis1/chTOG microtubule polymerase and the Mal3/EB1 microtubule plus-end tracking protein directly or indirectly bind Ndc80, thereby ensuring stable kinetochore-microtubule attachment. However, the purification of the Ndc80 complex from this yeast has not been achieved, which hampers the in-depth investigation as to how the outer kinetochore attaches to the plus end of the spindle microtubule. Here we report the two-step purification of the fission yeast Ndc80 holo complex from bacteria. First, we purified separately two sub-complexes consisting of Ndc80-Nuf2 and Spc24-Spc25. Then, these two sub-complexes were mixed and applied to size-exclusion chromatography. The reconstituted Ndc80 holo complex is composed of four subunits with equal stoichiometry. The complex possesses microtubule-binding activity, and Total Internal Reflection Fluorescence (TIRF)-microscopy assays show that the complex binds the microtubule lattice. Interestingly, unlike the human complex, the fission yeast complex does not track depolymerising microtubule ends. Further analysis shows that under physiological ionic conditions, the Ndc80 holo complex does not detectably bind Dis1, but instead it interacts with Mal3/EB1, by which the Ndc80 complex tracks the growing microtubule plus end. This result substantiates the notion that the Ndc80 complex plays a crucial role in establishment of the dynamic kinetochore-microtubule interface by cooperating with chTOG and EB1.


Assuntos
Regulação Fúngica da Expressão Gênica , Vetores Genéticos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Subunidades Proteicas/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Microscopia de Fluorescência/métodos , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/metabolismo , Schizosaccharomyces/ultraestrutura , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Proteínas de Schizosaccharomyces pombe/metabolismo
9.
Sci Rep ; 7(1): 1393, 2017 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-28469148

RESUMO

During Schizosaccharomyces pombe meiotic prophase, homologous chromosomes are co-aligned by linear elements (LinEs) analogous to the axial elements of the synaptonemal complex (SC) in other organisms. LinE proteins also promote the formation of meiotic DNA double-strand breaks (DSBs), the precursors of cross-overs. Rec10 is required for essentially all DSBs and recombination, and three others (Rec25, Rec27, and Mug20) are protein determinants of DSB hotspots - they bind DSB hotspots with high specificity and are required for DSB formation there. These four LinE proteins co-localize in the nucleus in an interdependent way, suggesting they form a complex. We used random mutagenesis to uncover recombination-deficient missense mutants with novel properties. Some missense mutations changed essential residues conserved among Schizosaccharomyces species. DSB formation, gene conversion, and crossing-over were coordinately reduced in the mutants tested. Based on our mutant analysis, we revised the rec27 open reading frame: the new start codon is in the previously annotated first intron. Genetic and fluorescence-microscopy assays indicated that the Rec10 N- and C-terminal regions have complex interactions with Rec25. These mutants are a valuable resource to elucidate further how LinE proteins and the related SCs of other species regulate meiotic DSB formation to form crossovers crucial for meiosis.


Assuntos
Proteínas de Ciclo Celular/genética , Quebras de DNA de Cadeia Dupla , Meiose , Proteínas Nucleares/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Ciclo Celular/isolamento & purificação , Conversão Gênica , Íntrons , Mutação de Sentido Incorreto , Proteínas Nucleares/isolamento & purificação , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/isolamento & purificação
10.
Biochem Biophys Res Commun ; 482(4): 896-901, 2017 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-27890612

RESUMO

Schizosaccharomyces pombe, which has a small genome but shares many physiological functions with higher eukaryotes, is a useful single-cell, model eukaryotic organism. In particular, many features concerning chromatin structure and dynamics, including heterochromatin, centromeres, telomeres, and DNA replication origins, are well conserved between S. pombe and higher eukaryotes. However, the S. pombe nucleosome, the fundamental structural unit of chromatin, has not been reconstituted in vitro. In the present study, we established the method to purify S. pombe histones H2A, H2B, H3, and H4, and successfully reconstituted the S. pombe nucleosome in vitro. Our thermal stability assay and micrococcal nuclease treatment assay revealed that the S. pombe nucleosome is markedly unstable and its DNA ends are quite accessible, as compared to the canonical human nucleosome. These findings are important to understand the mechanisms of epigenetic genomic DNA regulation in fission yeast.


Assuntos
Histonas/química , Nucleossomos/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/química , Sequência de Aminoácidos , DNA Fúngico/química , Histonas/isolamento & purificação , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Schizosaccharomyces/citologia , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Alinhamento de Sequência
11.
Acta Crystallogr F Struct Biol Commun ; 72(Pt 4): 263-8, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27050258

RESUMO

Dysfunction of histone-modifying enzymes affects chromatin regulation and is involved in carcinogenesis, tumour progression and other diseases. Histone methyltransferases are a family of key histone-modifying enzymes, but their structures, functions and mechanisms are incompletely understood, thus constraining drug-design efforts. Here, preliminary steps towards structure-function studies of Schizosaccharomyces pombe Set7, a putative histone methyltransferase and the first yeast full-length SET-domain-containing protein to be studied using X-ray crystallography, are reported. The methods from cloning to X-ray diffraction and phasing are discussed and the results will aid in prospective studies of histone-modifying enzymes.


Assuntos
Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Schizosaccharomyces/química , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Cristalização , Histona Metiltransferases , Histona-Lisina N-Metiltransferase , Proteínas de Schizosaccharomyces pombe/química
12.
Protein Expr Purif ; 97: 44-9, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24583182

RESUMO

Chromatin-associated proteins are heterogeneously and dynamically composed. To gain a complete understanding of DNA packaging and basic nuclear functions, it is important to generate a comprehensive inventory of these proteins. However, biochemical purification of chromatin-associated proteins is difficult and is accompanied by concerns over complex stability, protein solubility and yield. Here, we describe a new method for optimized purification of the endogenously expressed fission yeast Set2 complex, histone H3K36 methyltransferase. Using the standard centrifugation procedure for purification, approximately half of the Set2 protein separated into the insoluble chromatin pellet fraction, making it impossible to recover the large amounts of soluble Set2. To overcome this poor recovery, we developed a novel protein purification technique termed the filtration/immunoaffinity purification/mass spectrometry (FIM) method, which eliminates the need for centrifugation. Using the FIM method, in which whole cell lysates were filtered consecutively through eight different pore sizes (53-0.8µm), a high yield of soluble FLAG-tagged Set2 was obtained from fission yeast. The technique was suitable for affinity purification and produced a low background. A mass spectrometry analysis of anti-FLAG immunoprecipitated proteins revealed that Rpb1, Rpb2 and Rpb3, which have all been reported previously as components of the budding yeast Set2 complex, were isolated from fission yeast using the FIM method. In addition, other subunits of RNA polymerase II and its phosphatase were also identified. In conclusion, the FIM method is valid for the efficient purification of protein complexes that separate into the insoluble chromatin pellet fraction during centrifugation.


Assuntos
Histona-Lisina N-Metiltransferase/isolamento & purificação , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Cromatografia Líquida/métodos , Filtração/métodos , Immunoblotting/métodos , Imunoprecipitação/métodos , Espectrometria de Massas/métodos , Schizosaccharomyces/química
13.
J Cell Sci ; 127(Pt 11): 2460-70, 2014 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24652833

RESUMO

In yeasts, small intrinsically disordered proteins (IDPs) modulate ribonucleotide reductase (RNR) activity to ensure an optimal supply of dNTPs for DNA synthesis. The Schizosaccharomyces pombe Spd1 protein can directly inhibit the large RNR subunit (R1), import the small subunit (R2) into the nucleus and induce an architectural change in the R1-R2 holocomplex. Here, we report the characterization of Spd2, a protein with sequence similarity to Spd1. We show that Spd2 is a CRL4(Cdt2)-controlled IDP that functions together with Spd1 in the DNA damage response and in modulation of RNR architecture. However, Spd2 does not regulate dNTP pools and R2 nuclear import. Furthermore, deletion of spd2 only weakly suppresses the Rad3(ATR) checkpoint dependency of CRL4(Cdt2) mutants. However, when we raised intracellular dNTP pools by inactivation of RNR feedback inhibition, deletion of spd2 could suppress the checkpoint dependency of CRL4(Cdt2) mutant cells to the same extent as deletion of spd1. Collectively, these observations suggest that Spd1 on its own regulates dNTP pools, whereas in combination with Spd2 it modulates RNR architecture and sensitizes cells to DNA damage.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Ribonucleotídeo Redutases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação Alostérica/genética , Sequência de Aminoácidos , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Quinase do Ponto de Checagem 2/metabolismo , Reparo do DNA/genética , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/isolamento & purificação , Dados de Sequência Molecular , Mutação/genética , Nucleotidases/metabolismo , Conformação Proteica , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Homologia de Sequência de Aminoácidos
14.
Biochim Biophys Acta ; 1844(4): 767-77, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24382491

RESUMO

Translin is a single-stranded DNA and RNA binding protein that has a high affinity for G-rich sequences. TRAX is a Translin paralog that associates with Translin. Both Translin and TRAX were highly conserved in eukaryotes. The nucleic acid binding form of Translin is a barrel-shaped homo-octamer. A Translin-TRAX hetero-octamer having a similar structure also binds nucleic acids. Previous reports suggested that Translin may be involved in chromosomal translocations, telomere metabolism and the control of mRNA transport and translation. More recent studies have indicated that Translin-TRAX hetero-octamers are involved in RNA silencing. To gain a further insight into the functions of Translin, we have undertaken to systematically search for proteins with which it forms specific complexes in living cells. Here we report the results of such a search conducted in the fission yeast Schizosaccharomyces pombe, a suitable model system. This search was carried out by affinity purification and immuno-precipitation techniques, combined with differential labeling of the intracellular proteins with the stable isotopes ¹5N and ¹4N. We identified for the first time two proteins containing an RNA Recognition Motif (RRM), which are specifically associated with the yeast Translin: (1) the pre-mRNA-splicing factor srp1 that belongs to the highly conserved SR family of proteins and (2) vip1, a protein conserved in fungi. Our data also support the presence of RNA in these intracellular complexes. Our experimental approach should be generally applicable to studies of weak intracellular protein-protein interactions and provides a clear distinction between false positive vs. truly interacting proteins.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , DNA Fúngico/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , RNA Fúngico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , DNA Fúngico/química , Fosfotransferases (Aceptor do Grupo Fosfato)/química , Fosfotransferases (Aceptor do Grupo Fosfato)/isolamento & purificação , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Fatores de Processamento de RNA , RNA Fúngico/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/isolamento & purificação , Schizosaccharomyces/química , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/isolamento & purificação
15.
J Biol Chem ; 289(5): 2725-35, 2014 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-24327658

RESUMO

Sterol homeostasis is tightly controlled by the sterol regulatory element-binding protein (SREBP) transcription factor that is highly conserved from fungi to mammals. In fission yeast, SREBP functions in an oxygen-sensing pathway to promote adaptation to decreased oxygen supply that limits oxygen-dependent sterol synthesis. Low oxygen stimulates proteolytic cleavage of the SREBP homolog Sre1, generating the active transcription factor Sre1N that drives expression of sterol biosynthetic enzymes. In addition, low oxygen increases the stability and DNA binding activity of Sre1N. To identify additional signals controlling Sre1 activity, we conducted a genetic overexpression screen. Here, we describe our isolation and characterization of the casein kinase 1 family member Hhp2 as a novel regulator of Sre1N. Deletion of Hhp2 increases Sre1N protein stability and ergosterol levels in the presence of oxygen. Hhp2-dependent Sre1N degradation by the proteasome requires Hhp2 kinase activity, and Hhp2 binds and phosphorylates Sre1N at specific residues. Our results describe a role for casein kinase 1 as a direct regulator of sterol homeostasis. Given the role of mammalian Hhp2 homologs, casein kinase 1δ and 1ε, in regulation of the circadian clock, these findings may provide a mechanism for coordinating circadian rhythm and lipid metabolism.


Assuntos
Caseína Quinase I/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Esteróis/metabolismo , Caseína Quinase I/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Homeostase/fisiologia , Metabolismo dos Lipídeos/fisiologia , Oxigênio/metabolismo , Fosforilação/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/isolamento & purificação , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Proteínas de Ligação a Elemento Regulador de Esterol/genética
16.
BMC Biochem ; 14: 5, 2013 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-23444842

RESUMO

BACKGROUND: The hetero-hexamer of the eukaryotic minichromosome maintenance (MCM) proteins plays an essential role in replication of genomic DNA. The ring-shaped Mcm2-7 hexamers comprising one of each subunit show helicase activity in vitro, and form double-hexamers on DNA. The Mcm4/6/7 also forms a hexameric complex with helicase activity in vitro. RESULTS: We used an Escherichiai coli expression system to express various domains of Schizosaccharomyces pombe Mcm4, 6 and 7 in order to characterize their domain structure, oligomeric states, and possible inter-/intra-subunit interactions. We also successfully employed a co-expression system to express Mcm4/6/7 at the same time in Escherichiai coli, and have purified functional Mcm4/6/7 complex in a hexameric state in high yield and purity, providing a means for generating large quantity of proteins for future structural and biochemical studies. CONCLUSIONS: Based on our results and those of others, models were proposed for the subunit arrangement and architecture of both the Mcm4/6/7 hexamer and the Mcm2-7 double-hexamer.


Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Componente 4 do Complexo de Manutenção de Minicromossomo , Componente 6 do Complexo de Manutenção de Minicromossomo , Componente 7 do Complexo de Manutenção de Minicromossomo , Ligação Proteica , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Proteínas de Schizosaccharomyces pombe/metabolismo
17.
Protein Expr Purif ; 88(2): 207-13, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23337086

RESUMO

During meiosis, chromosomes adopt a bouquet arrangement, which is widely conserved among eukaryotes. This arrangement is assumed to play an important role in the normal progression of meiosis, by mediating the proper pairing of homologous chromosomes. In Schizosaccharomyces pombe, the complex of Bqt1 and Bqt2 plays a key role in telomere clustering and the subsequent bouquet arrangement of chromosomes during early meiotic prophase. Bqt1 and Bqt2 are part of a multi-protein complex that mediates the attachment of the telomere to the nuclear membrane. However, the structural details of the complex are needed to clarify the mechanism of telomere clustering. To enable biophysical studies of Bqt1 and Bqt2, we established a purification procedure for the Schizosaccharomyces japonicus Bqt1-Bqt2 complex, which is closely related to the S. pombe Bqt1-Bqt2 complex. A co-expression vector, in which one of the expressed subunits is fused to a removable SUMO tag, yielded high amounts of the proteins in the soluble fraction. The solubility of the Bqt1-Bqt2 complex after the removal of the SUMO tag was maintained by including CHAPS, a nondenaturing, zwitterionic detergent, in the purification buffers. These procedures enabled us to rapidly purify the stable Bqt1-Bqt2 complex. The co-purified Bqt1 and Bqt2 proteins formed a stable heterodimer, consistent with results from in vivo studies showing the requirement of both proteins for the bouquet arrangement. The expression and purification procedures established here will facilitate further biophysical studies of the Bqt1-Bqt2 complex.


Assuntos
Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Schizosaccharomyces/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/isolamento & purificação , Sequência de Aminoácidos , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos/genética , Dados de Sequência Molecular , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Schizosaccharomyces/química , Proteínas de Schizosaccharomyces pombe/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/isolamento & purificação , Solubilidade , Proteínas de Ligação a Telômeros/química
18.
FEBS Lett ; 585(24): 3850-5, 2011 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-22036784

RESUMO

The MCM (mini-chromosome maintenance) complex is the core of the eukaryotic replicative helicase and comprises six proteins, Mcm2-Mcm7. In humans, a variant form of the complex has Mcm2 replaced by the MCM-BP protein. Recent results suggest that a similar complex exists in fission yeast with an essential role in DNA replication and cell cycle progression. Here, we describe the purification and subunit composition of the fission yeast MCM(Mcb1) complex. Using newly generated temperature-sensitive alleles, we show that loss of MCM(Mcb1) function leads to accumulation of DNA damage, checkpoint activation and cell cycle arrest, and provide evidence for a role for MCM(Mcb1) in meiosis.


Assuntos
Complexos Multiproteicos/isolamento & purificação , Complexos Multiproteicos/metabolismo , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Quinase 1 do Ponto de Checagem , Cromatografia de Afinidade , Cromossomos Fúngicos/genética , Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA Fúngico/biossíntese , DNA Fúngico/genética , Proteínas de Ligação a DNA/metabolismo , Espectrometria de Massas , Meiose , Complexos Multiproteicos/análise , Complexos Multiproteicos/química , Proteínas Quinases/metabolismo , Subunidades Proteicas/análise , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/análise , Proteínas de Schizosaccharomyces pombe/química
19.
Methods Mol Biol ; 725: 1-13, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21528443

RESUMO

Small interfering (si) RNAs, produced by the RNA interference (RNAi)-mediated processing of long double-stranded (ds) RNAs, can inhibit gene expression by post-transcriptional or transcriptional gene silencing mechanisms. At the heart of all small RNA-mediated silencing lies the key RNAi effector protein Argonaute, which once loaded with small RNAs can recognize its target transcript by siRNA-RNA Watson-Crick base pairing interactions. In the fission yeast Schizosaccharomyces pombe, the formation of the epigenetically heritable centromeric heterochromatin requires RNAi proteins including the sole fission yeast Argonaute homolog, Ago1. Two distinct native Ago1 complexes have been purified and studied extensively, both of which are required for siRNA production and heterochromatin formation at the fission yeast centromeres. The purification and analysis of the Argonaute siRNA chaperone (ARC) complex and RNA-induced transcriptional silencing (RITS) complex have provided insight into the mechanism of siRNA-Ago1 loading and the cis recruitment of silencing complexes at fission yeast centromeres, respectively. These discoveries have been instrumental in shaping the current models of RNA-mediated epigenetic silencing in eukaryotes. Below, we describe the protocol used for affinity purification of the native Ago1 complexes from S. pombe.


Assuntos
Biotecnologia/métodos , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas de Ligação a RNA/metabolismo , Complexo de Inativação Induzido por RNA/isolamento & purificação , Complexo de Inativação Induzido por RNA/metabolismo , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas Argonautas , Western Blotting , Cáusticos/farmacologia , Precipitação Química/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Reação em Cadeia da Polimerase , Schizosaccharomyces/crescimento & desenvolvimento , Coloração pela Prata , Transformação Genética , Ácido Tricloroacético/farmacologia
20.
J Biol Chem ; 286(7): 5784-92, 2011 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-21148484

RESUMO

Fission yeast Schizosaccharomyces pombe is an important genetic model organism for studying the mechanisms of endocytosis and cytokinesis. However, most work on the biochemical properties of fission yeast actin-binding proteins has been done with skeletal muscle actin for matters of convenience. When simulations of mathematical models of the mechanism of endocytosis were compared with events in live cells, some of the reactions appeared to be much faster than observed in biochemical experiments with muscle actin. Here, we used gelsolin affinity chromatography to purify actin from fission yeast. S. pombe actin shares many properties with skeletal muscle actin but has higher intrinsic nucleotide exchange rate, faster trimer nucleus formation, faster phosphate dissociation rate from polymerized actin, and faster nucleation of actin filaments with Arp2/3 complex. These properties close the gap between the biochemistry and predictions made by mathematical models of endocytosis in S. pombe cells.


Assuntos
Actinas/química , Actinas/isolamento & purificação , Modelos Biológicos , Músculo Esquelético/química , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Schizosaccharomyces/química , Complexo 2-3 de Proteínas Relacionadas à Actina/química , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Gelsolina/química , Camundongos , Músculo Esquelético/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo
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