Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 23.901
Filtrar
1.
Nat Commun ; 11(1): 4905, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32999288

RESUMO

Transcription factor (TF) IIIC is a conserved eukaryotic six-subunit protein complex with dual function. It serves as a general TF for most RNA polymerase (Pol) III genes by recruiting TFIIIB, but it is also involved in chromatin organization and regulation of Pol II genes through interaction with CTCF and condensin II. Here, we report the structure of the S. cerevisiae TFIIIC subcomplex τA, which contains the most conserved subunits of TFIIIC and is responsible for recruitment of TFIIIB and transcription start site (TSS) selection at Pol III genes. We show that τA binding to its promoter is auto-inhibited by a disordered acidic tail of subunit τ95. We further provide a negative-stain reconstruction of τA bound to the TFIIIB subunits Brf1 and TBP. This shows that a ruler element in τA achieves positioning of TFIIIB upstream of the TSS, and suggests remodeling of the complex during assembly of TFIIIB by TFIIIC.


Assuntos
Regulação Fúngica da Expressão Gênica , RNA Polimerase III/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae/genética , Fatores de Transcrição TFIII/ultraestrutura , Animais , Linhagem Celular , Microscopia Crioeletrônica , DNA Fúngico/genética , DNA Fúngico/metabolismo , Genes Fúngicos/genética , Insetos , Domínios Proteicos , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIIB/genética , Fator de Transcrição TFIIIB/isolamento & purificação , Fator de Transcrição TFIIIB/metabolismo , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFIII/isolamento & purificação , Fatores de Transcrição TFIII/metabolismo , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética
2.
PLoS Comput Biol ; 16(9): e1007815, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32925900

RESUMO

Protein-protein interactions are involved in a wide range of cellular processes. These interactions often involve intrinsically disordered proteins (IDPs) and protein binding domains. However, the details of IDP binding pathways are hard to characterize using experimental approaches, which can rarely capture intermediate states present at low populations. SH3 domains are common protein interaction domains that typically bind proline-rich disordered segments and are involved in cell signaling, regulation, and assembly. We hypothesized, given the flexibility of SH3 binding peptides, that their binding pathways include multiple steps important for function. Molecular dynamics simulations were used to characterize the steps of binding between the yeast Abp1p SH3 domain (AbpSH3) and a proline-rich IDP, ArkA. Before binding, the N-terminal segment 1 of ArkA is pre-structured and adopts a polyproline II helix, while segment 2 of ArkA (C-terminal) adopts a 310 helix, but is far less structured than segment 1. As segment 2 interacts with AbpSH3, it becomes more structured, but retains flexibility even in the fully engaged state. Binding simulations reveal that ArkA enters a flexible encounter complex before forming the fully engaged bound complex. In the encounter complex, transient nonspecific hydrophobic and long-range electrostatic contacts form between ArkA and the binding surface of SH3. The encounter complex ensemble includes conformations with segment 1 in both the forward and reverse orientation, suggesting that segment 2 may play a role in stabilizing the correct binding orientation. While the encounter complex forms quickly, the slow step of binding is the transition from the disordered encounter ensemble to the fully engaged state. In this transition, ArkA makes specific contacts with AbpSH3 and buries more hydrophobic surface. Simulating the binding between ApbSH3 and ArkA provides insight into the role of encounter complex intermediates and nonnative hydrophobic interactions for other SH3 domains and IDPs in general.


Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas dos Microfilamentos , Proteínas de Saccharomyces cerevisiae , Domínios de Homologia de src , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Domínios de Homologia de src/genética , Domínios de Homologia de src/fisiologia
3.
Mol Genet Genomics ; 295(6): 1489-1500, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32948893

RESUMO

Glucose, fructose and mannose are the preferred carbon/energy sources for the yeast Saccharomyces cerevisiae. Absence of preferred energy sources activates glucose derepression, which is regulated by the kinase Snf1. Snf1 phosphorylates the transcriptional repressor Mig1, which results in its exit from the nucleus and subsequent derepression of genes. In contrast, Snf1 is inactive when preferred carbon sources are available, which leads to dephosphorylation of Mig1 and its translocation to the nucleus where Mig1 acts as a transcription repressor. Here we revisit the role of the three hexose kinases, Hxk1, Hxk2 and Glk1, in glucose de/repression. We demonstrate that all three sugar kinases initially affect Mig1 nuclear localization upon addition of glucose, fructose and mannose. This initial import of Mig1 into the nucleus was temporary; for continuous nucleocytoplasmic shuttling of Mig1, Hxk2 is required in the presence of glucose and mannose and in the presence of fructose Hxk2 or Hxk1 is required. Our data suggest that Mig1 import following exposure to preferred energy sources is controlled via two different pathways, where (1) the initial import is regulated by signals derived from metabolism and (2) continuous shuttling is regulated by the Hxk2 and Hxk1 proteins. Mig1 nucleocytoplasmic shuttling appears to be important for the maintenance of the repressed state in which Hxk1/2 seems to play an essential role.


Assuntos
Núcleo Celular/metabolismo , Frutose/metabolismo , Glucose/metabolismo , Hexoquinase/metabolismo , Manose/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Regulação Fúngica da Expressão Gênica , Hexoquinase/genética , Fosforilação , Transporte Proteico , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
4.
Mol Cell ; 80(1): 72-86.e7, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32910895

RESUMO

Membrane protein biogenesis faces the challenge of chaperoning hydrophobic transmembrane helices for faithful membrane insertion. The guided entry of tail-anchored proteins (GET) pathway targets and inserts tail-anchored (TA) proteins into the endoplasmic reticulum (ER) membrane with an insertase (yeast Get1/Get2 or mammalian WRB/CAML) that captures the TA from a cytoplasmic chaperone (Get3 or TRC40, respectively). Here, we present cryo-electron microscopy reconstructions, native mass spectrometry, and structure-based mutagenesis of human WRB/CAML/TRC40 and yeast Get1/Get2/Get3 complexes. Get3 binding to the membrane insertase supports heterotetramer formation, and phosphatidylinositol binding at the heterotetramer interface stabilizes the insertase for efficient TA insertion in vivo. We identify a Get2/CAML cytoplasmic helix that forms a "gating" interaction with Get3/TRC40 important for TA insertion. Structural homology with YidC and the ER membrane protein complex (EMC) implicates an evolutionarily conserved insertion mechanism for divergent substrates utilizing a hydrophilic groove. Thus, we provide a detailed structural and mechanistic framework to understand TA membrane insertion.


Assuntos
Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Complexos Multiproteicos/metabolismo , Linhagem Celular , Sequência Conservada , Evolução Molecular , Humanos , Proteínas de Membrana/metabolismo , Modelos Moleculares , Fosfatidilinositóis/metabolismo , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Mol Cell ; 80(1): 114-126.e8, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32916094

RESUMO

DNA replication is carried out by a multi-protein machine called the replisome. In Saccharomyces cerevisiae, the replisome is composed of over 30 different proteins arranged into multiple subassemblies, each performing distinct activities. Synchrony of these activities is required for efficient replication and preservation of genomic integrity. How this is achieved is particularly puzzling at the lagging strand, where current models of the replisome architecture propose turnover of the canonical lagging strand polymerase, Pol δ, at every cycle of Okazaki fragment synthesis. Here, we established single-molecule fluorescence microscopy protocols to study the binding kinetics of individual replisome subunits in live S. cerevisiae. Our results show long residence times for most subunits at the active replisome, supporting a model where all subassemblies bind tightly and work in a coordinated manner for extended periods, including Pol δ, redefining the architecture of the active eukaryotic replisome.


Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Células Eucarióticas/metabolismo , Complexos Multienzimáticos/metabolismo , Núcleo Celular/metabolismo , Cinética , Modelos Biológicos , Proteínas Nucleares/metabolismo , Subunidades Proteicas/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula , Fatores de Tempo
6.
Nat Commun ; 11(1): 4625, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934225

RESUMO

A hallmark of neurodegeneration is defective protein quality control. The E3 ligase Listerin (LTN1/Ltn1) acts in a specialized protein quality control pathway-Ribosome-associated Quality Control (RQC)-by mediating proteolytic targeting of incomplete polypeptides produced by ribosome stalling, and Ltn1 mutation leads to neurodegeneration in mice. Whether neurodegeneration results from defective RQC and whether defective RQC contributes to human disease have remained unknown. Here we show that three independently-generated mouse models with mutations in a different component of the RQC complex, NEMF/Rqc2, develop progressive motor neuron degeneration. Equivalent mutations in yeast Rqc2 selectively interfere with its ability to modify aberrant translation products with C-terminal tails which assist with RQC-mediated protein degradation, suggesting a pathomechanism. Finally, we identify NEMF mutations expected to interfere with function in patients from seven families presenting juvenile neuromuscular disease. These uncover NEMF's role in translational homeostasis in the nervous system and implicate RQC dysfunction in causing neurodegeneration.


Assuntos
Doenças Neuromusculares/metabolismo , Ribossomos/metabolismo , Sequência de Aminoácidos , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Doenças Neuromusculares/genética , Doenças Neuromusculares/patologia , Proteólise , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência
7.
PLoS Genet ; 16(8): e1008569, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32810145

RESUMO

Correct bioriented attachment of sister chromatids to the mitotic spindle is essential for chromosome segregation. In budding yeast, the conserved protein shugoshin (Sgo1) contributes to biorientation by recruiting the protein phosphatase PP2A-Rts1 and the condensin complex to centromeres. Using peptide prints, we identified a Serine-Rich Motif (SRM) of Sgo1 that mediates the interaction with condensin and is essential for centromeric condensin recruitment and the establishment of biorientation. We show that the interaction is regulated via phosphorylation within the SRM and we determined the phospho-sites using mass spectrometry. Analysis of the phosphomimic and phosphoresistant mutants revealed that SRM phosphorylation disrupts the shugoshin-condensin interaction. We present evidence that Mps1, a central kinase in the spindle assembly checkpoint, directly phosphorylates Sgo1 within the SRM to regulate the interaction with condensin and thereby condensin localization to centromeres. Our findings identify novel mechanisms that control shugoshin activity at the centromere in budding yeast.


Assuntos
Adenosina Trifosfatases/metabolismo , Centrômero/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
8.
PLoS Genet ; 16(8): e1008745, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32845888

RESUMO

Sphingolipids are abundant and essential molecules in eukaryotes that have crucial functions as signaling molecules and as membrane components. Sphingolipid biosynthesis starts in the endoplasmic reticulum with the condensation of serine and palmitoyl-CoA. Sphingolipid biosynthesis is highly regulated to maintain sphingolipid homeostasis. Even though, serine is an essential component of the sphingolipid biosynthesis pathway, its role in maintaining sphingolipid homeostasis has not been precisely studied. Here we show that serine uptake is an important factor for the regulation of sphingolipid biosynthesis in Saccharomyces cerevisiae. Using genetic experiments, we find the broad-specificity amino acid permease Gnp1 to be important for serine uptake. We confirm these results with serine uptake assays in gnp1Δ cells. We further show that uptake of exogenous serine by Gnp1 is important to maintain cellular serine levels and observe a specific connection between serine uptake and the first step of sphingolipid biosynthesis. Using mass spectrometry-based flux analysis, we further observed imported serine as the main source for de novo sphingolipid biosynthesis. Our results demonstrate that yeast cells preferentially use the uptake of exogenous serine to regulate sphingolipid biosynthesis. Our study can also be a starting point to analyze the role of serine uptake in mammalian sphingolipid metabolism.


Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismo , Esfingolipídeos/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Homeostase , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Esfingolipídeos/biossíntese
9.
BMC Bioinformatics ; 21(1): 355, 2020 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-32787776

RESUMO

BACKGROUND: The accurate annotation of protein functions is of great significance in elucidating the phenomena of life, treating disease and developing new medicines. Various methods have been developed to facilitate the prediction of these functions by combining protein interaction networks (PINs) with multi-omics data. However, it is still challenging to make full use of multiple biological to improve the performance of functions annotation. RESULTS: We presented NPF (Network Propagation for Functions prediction), an integrative protein function predicting framework assisted by network propagation and functional module detection, for discovering interacting partners with similar functions to target proteins. NPF leverages knowledge of the protein interaction network architecture and multi-omics data, such as domain annotation and protein complex information, to augment protein-protein functional similarity in a propagation manner. We have verified the great potential of NPF for accurately inferring protein functions. According to the comprehensive evaluation of NPF, it delivered a better performance than other competing methods in terms of leave-one-out cross-validation and ten-fold cross validation. CONCLUSIONS: We demonstrated that network propagation, together with multi-omics data, can both discover more partners with similar function, and is unconstricted by the "small-world" feature of protein interaction networks. We conclude that the performance of function prediction depends greatly on whether we can extract and exploit proper functional information of similarity from protein correlations.


Assuntos
Algoritmos , Biologia Computacional/métodos , Mapas de Interação de Proteínas , Análise por Conglomerados , Ontologia Genética , Ligação Proteica , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
Nat Commun ; 11(1): 4281, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855416

RESUMO

Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is crucial for regulating cellular energy metabolism. Conformational transitions of the transcription initiation complex must be central for such control, but how the conformational dynamics progress throughout transcription initiation remains unknown. Here, we use single-molecule fluorescence resonance energy transfer techniques to examine the conformational dynamics of the transcriptional system of yeast mitochondria with single-base resolution. We show that the yeast mitochondrial transcriptional complex dynamically transitions among closed, open, and scrunched states throughout the initiation stage. Then abruptly at position +8, the dynamic states of initiation make a sharp irreversible transition to an unbent conformation with associated promoter release. Remarkably, stalled initiation complexes remain in dynamic scrunching and unscrunching states without dissociating the RNA transcript, implying the existence of backtracking transitions with possible regulatory roles. The dynamic landscape of transcription initiation suggests a kinetically driven regulation of mitochondrial transcription.


Assuntos
Mitocôndrias/genética , Saccharomyces cerevisiae/genética , Iniciação da Transcrição Genética , Trifosfato de Adenosina , DNA Fúngico/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula/métodos , Elongação da Transcrição Genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
11.
Nat Commun ; 11(1): 3907, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764578

RESUMO

Nucleic acids can fold into G-quadruplex (G4) structures that can fine-tune biological processes. Proteins are required to recognize G4 structures and coordinate their function. Here we identify Zuo1 as a novel G4-binding protein in vitro and in vivo. In vivo in the absence of Zuo1 fewer G4 structures form, cell growth slows and cells become UV sensitive. Subsequent experiments reveal that these cellular changes are due to reduced levels of G4 structures. Zuo1 function at G4 structures results in the recruitment of nucleotide excision repair (NER) factors, which has a positive effect on genome stability. Cells lacking functional NER, as well as Zuo1, accumulate G4 structures, which become accessible to translesion synthesis. Our results suggest a model in which Zuo1 supports NER function and regulates the choice of the DNA repair pathway nearby G4 structures.


Assuntos
Reparo do DNA/fisiologia , Quadruplex G , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sítios de Ligação/genética , Dano ao DNA , Reparo do DNA/genética , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , Deleção de Genes , Aptidão Genética , Genoma Fúngico , Instabilidade Genômica , Modelos Biológicos , Chaperonas Moleculares/genética , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
12.
Nat Commun ; 11(1): 4154, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32814778

RESUMO

The DNA damage response (DDR) coordinates DNA metabolism with nuclear and non-nuclear processes. The DDR kinase Rad53CHK1/CHK2 controls histone degradation to assist DNA repair. However, Rad53 deficiency causes histone-dependent growth defects in the absence of DNA damage, pointing out unknown physiological functions of the Rad53-histone axis. Here we show that histone dosage control by Rad53 ensures metabolic homeostasis. Under physiological conditions, Rad53 regulates histone levels through inhibitory phosphorylation of the transcription factor Spt21NPAT on Ser276. Rad53-Spt21 mutants display severe glucose dependence, caused by excess histones through two separable mechanisms: dampening of acetyl-coenzyme A-dependent carbon metabolism through histone hyper-acetylation, and Sirtuin-mediated silencing of starvation-induced subtelomeric domains. We further demonstrate that repression of subtelomere silencing by physiological Tel1ATM and Rpd3HDAC activities coveys tolerance to glucose restriction. Our findings identify DDR mutations, histone imbalances and aberrant subtelomeric chromatin as interconnected causes of glucose dependence, implying that DDR kinases coordinate metabolism and epigenetic changes.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Glucose/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , Dano ao DNA , Reparo do DNA , Inativação Gênica , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Serina/genética , Serina/metabolismo , Telômero/genética , Fatores de Transcrição/genética
13.
Nat Commun ; 11(1): 4206, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826896

RESUMO

Saccharomyces cerevisiae TBP associated factor 14 (Taf14) is a well-studied transcriptional regulator that controls diverse physiological processes and that physically interacts with at least seven nuclear complexes in yeast. Despite multiple previous Taf14 structural studies, the nature of its disparate transcriptional regulatory functions remains opaque. Here, we demonstrate that the extra-terminal (ET) domain of Taf14 (Taf14ET) recognizes a common motif in multiple transcriptional coactivator proteins from several nuclear complexes, including RSC, SWI/SNF, INO80, NuA3, TFIID, and TFIIF. Moreover, we show that such partner binding promotes liquid-liquid phase separation (LLPS) of Taf14ET, in a mechanism common to YEATS-associated ET domains (e.g., AF9ET) but not Bromo-associated ET domains from BET-family proteins. Thus, beyond identifying the molecular mechanism by which Taf14ET associates with many transcriptional regulators, our study suggests that Taf14 may function as a versatile nuclear hub that orchestrates transcriptional machineries to spatiotemporally regulate diverse cellular pathways.


Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIID/metabolismo , Proteínas de Transporte , Proteínas de Ciclo Celular/metabolismo , Análise por Conglomerados , Proteínas de Ligação a DNA , Epigenômica , Regulação Fúngica da Expressão Gênica , Modelos Moleculares , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/genética , Fatores de Transcrição/metabolismo
14.
Nat Commun ; 11(1): 4263, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848132

RESUMO

Eukaryotic DNA replication initiation relies on the origin recognition complex (ORC), a DNA-binding ATPase that loads the Mcm2-7 replicative helicase onto replication origins. Here, we report cryo-electron microscopy (cryo-EM) structures of DNA-bound Drosophila ORC with and without the co-loader Cdc6. These structures reveal that Orc1 and Orc4 constitute the primary DNA binding site in the ORC ring and cooperate with the winged-helix domains to stabilize DNA bending. A loop region near the catalytic Walker B motif of Orc1 directly contacts DNA, allosterically coupling DNA binding to ORC's ATPase site. Correlating structural and biochemical data show that DNA sequence modulates DNA binding and remodeling by ORC, and that DNA bending promotes Mcm2-7 loading in vitro. Together, these findings explain the distinct DNA sequence-dependencies of metazoan and S. cerevisiae initiators in origin recognition and support a model in which DNA geometry and bendability contribute to Mcm2-7 loading site selection in metazoans.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/metabolismo , Origem de Replicação , Domínio AAA , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Microscopia Crioeletrônica , DNA/química , DNA/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Hidrólise , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Modelos Moleculares , Complexo de Reconhecimento de Origem/genética , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Origem de Replicação/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
PLoS Biol ; 18(8): e3000836, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32804946

RESUMO

Pleiotropy-when a single mutation affects multiple traits-is a controversial topic with far-reaching implications. Pleiotropy plays a central role in debates about how complex traits evolve and whether biological systems are modular or are organized such that every gene has the potential to affect many traits. Pleiotropy is also critical to initiatives in evolutionary medicine that seek to trap infectious microbes or tumors by selecting for mutations that encourage growth in some conditions at the expense of others. Research in these fields, and others, would benefit from understanding the extent to which pleiotropy reflects inherent relationships among phenotypes that correlate no matter the perturbation (vertical pleiotropy). Alternatively, pleiotropy may result from genetic changes that impose correlations between otherwise independent traits (horizontal pleiotropy). We distinguish these possibilities by using clonal populations of yeast cells to quantify the inherent relationships between single-cell morphological features. Then, we demonstrate how often these relationships underlie vertical pleiotropy and how often these relationships are modified by genetic variants (quantitative trait loci [QTL]) acting via horizontal pleiotropy. Our comprehensive screen measures thousands of pairwise trait correlations across hundreds of thousands of yeast cells and reveals ample evidence of both vertical and horizontal pleiotropy. Additionally, we observe that the correlations between traits can change with the environment, genetic background, and cell-cycle position. These changing dependencies suggest a nuanced view of pleiotropy: biological systems demonstrate limited pleiotropy in any given context, but across contexts (e.g., across diverse environments and genetic backgrounds) each genetic change has the potential to influence a larger number of traits. Our method suggests that exploiting pleiotropy for applications in evolutionary medicine would benefit from focusing on traits with correlations that are less dependent on context.


Assuntos
Pleiotropia Genética , Modelos Genéticos , Herança Multifatorial , Locos de Características Quantitativas , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Evolução Biológica , Ciclo Celular/genética , Células Clonais , Variação Genética , Ensaios de Triagem em Larga Escala , Mutação , Fenótipo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Célula Única
16.
PLoS Comput Biol ; 16(8): e1008145, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32853212

RESUMO

Oligomeric proteins are central to life. Duplication and divergence of their genes is a key evolutionary driver, also because duplications can yield very different outcomes. Given a homomeric ancestor, duplication can yield two paralogs that form two distinct homomeric complexes, or a heteromeric complex comprising both paralogs. Alternatively, one paralog remains a homomer while the other acquires a new partner. However, so far, conflicting trends have been noted with respect to which fate dominates, primarily because different methods and criteria are being used to assign the interaction status of paralogs. Here, we systematically analyzed all Saccharomyces cerevisiae and Escherichia coli oligomeric complexes that include paralogous proteins. We found that the proportions of homo-hetero duplication fates strongly depend on a variety of factors, yet that nonetheless, rigorous filtering gives a consistent picture. In E. coli about 50%, of the paralogous pairs appear to have retained the ancestral homomeric interaction, whereas in S. cerevisiae only ~10% retained a homomeric state. This difference was also observed when unique complexes were counted instead of paralogous gene pairs. We further show that this difference is accounted for by multiple cases of heteromeric yeast complexes that share common ancestry with homomeric bacterial complexes. Our analysis settles contradicting trends and conflicting previous analyses, and provides a systematic and rigorous pipeline for delineating the fate of duplicated oligomers in any organism for which protein-protein interaction data are available.


Assuntos
Evolução Biológica , Proteínas de Escherichia coli/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Escherichia coli/genética , Duplicação Gênica , Proteínas de Saccharomyces cerevisiae/genética
17.
PLoS One ; 15(8): e0237982, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32817700

RESUMO

Some organisms, like Trichomonas vaginalis, contain mitochondria-related hydrogen-producing organelles, called hydrogenosomes. The protein targeting into these organelles is proposed to be similar to the well-studied mitochondria import. Indeed, S. cerevisiae mitochondria and T. vaginalis hydrogenosomes share some components of protein import complexes. However, it is still unknown whether targeting signals directing substrate proteins to hydrogenosomes can support in other eukaryotes specific mitochondrial localization. To address this issue, we investigated the intracellular localization of three hydrogenosomal tail-anchored proteins expressed in yeast cells. We observed that these proteins were targeted to both mitochondria and ER with a variable dependency on the mitochondrial MIM complex. Our results suggest that the targeting signal of TA proteins are only partially conserved between hydrogenosomes and yeast mitochondria.


Assuntos
Retículo Endoplasmático/metabolismo , Regulação Fúngica da Expressão Gênica , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Trichomonas vaginalis/citologia , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trichomonas vaginalis/genética , Trichomonas vaginalis/metabolismo
18.
Mol Cell ; 79(5): 768-781.e7, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32738194

RESUMO

Misfolded proteins in the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD). Although ERAD components involved in degradation of luminal substrates are well characterized, much less is known about quality control of membrane proteins. Here, we analyzed the degradation pathways of two short-lived ER membrane model proteins in mammalian cells. Using a CRISPR-Cas9 genome-wide library screen, we identified an ERAD branch required for quality control of a subset of membrane proteins. Using biochemical and mass spectrometry approaches, we showed that this ERAD branch is defined by an ER membrane complex consisting of the ubiquitin ligase RNF185, the ubiquitin-like domain containing proteins TMUB1/2 and TMEM259/Membralin, a poorly characterized protein. This complex cooperates with cytosolic ubiquitin ligase UBE3C and p97 ATPase in degrading their membrane substrates. Our data reveal that ERAD branches have remarkable specificity for their membrane substrates, suggesting that multiple, perhaps combinatorial, determinants are involved in substrate selection.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Sistema Enzimático do Citocromo P-450/metabolismo , Células HEK293 , Células HeLa , Humanos , Domínios Proteicos , Dobramento de Proteína , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Esterol 14-Desmetilase/metabolismo
19.
PLoS One ; 15(8): e0236293, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32760074

RESUMO

To divide replicated chromosomes equally between daughter cells, kinetochores must attach to microtubules emanating from opposite poles of the mitotic spindle (biorientation). An error correction mechanism facilitates this process by destabilizing erroneous kinetochore-microtubule attachments. Here we present a stochastic model of kinetochore-microtubule attachments, via an essential protein Ndc80 in budding yeast, Saccharomyces cerevisiae. Using the model, we calculate the stochastic dynamics of a pair of sister kinetochores as they transition among different attachment states. First of all, we determine the kinase-to-phosphatase balance point that maximizes the probability of biorientation, while starting from an erroneous attachment state. We find that the balance point is sensitive to the rates of microtubule-Ndc80 dissociation and derive an approximate analytical formula that defines the balance point. Secondly, we determine the probability of transition from low-tension amphitelic to monotelic attachment and find that, despite this probability being approximately 33%, biorientation can be achieved with high probability. Thirdly, we calculate the contribution of the geometrical orientation of sister kinetochores to the probability of biorientation and show that, in the absence of geometrical orientation, the biorientation error rate is much larger than that observed in experiments. Finally, we study the coupling of the error correction mechanism to the spindle assembly checkpoint by calculating the average binding of checkpoint-related proteins to the kinetochore during the error correction process.


Assuntos
Segregação de Cromossomos , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Modelos Genéticos , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Pontos de Checagem da Fase M do Ciclo Celular/genética , Processos Estocásticos
20.
PLoS One ; 15(8): e0237540, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32804965

RESUMO

The yeast MAP kinase Hog1 pathway activates transcription of several hundreds genes. Large-scale gene expression and DNA binding assays suggest that most Hog1-induced genes are regulated by the transcriptional activators Msn2/4, Hot1 and Sko1. These studies also revealed the target genes of each activator and the putative binding sites on their promoters. In a previous study we identified a group of genes, which we considered the bona fide targets of Hog1, because they were induced in response to expression of intrinsically active mutant of Hog1, in the absence of any stress. We previously analyzed the promoter of the most highly induced gene, STL1, and noticed that some promoter properties were different from those proposed by large-scale data. We therefore continue to study promoters individually and present here analyses of promoters of more Hog1's targets, RTC3, HSP12, DAK1 and ALD3. We report that RTC3 and HSP12 promoters are robust and are induced, to different degrees, even in cells lacking all four activators. DAK1 and ALD3 promoters are not robust and fully depend on a single activator, DAK1 on Sko1 and ALD3 on Msn2/4. Most of these observations could not be inferred from the large-scale data. Msn2/4 are involved in regulating all four promoters. It was assumed, therefore, that the promoters are spontaneously active in ras2Δ cells, in which Msn2/4 are known to be de-repressed. Intriguingly, the promoters were not active in BY4741ras2Δ cells, but were de-repressed, as expected, in ras2Δ cells of other genetic backgrounds. This study describes two phenomena. One, some Hog1's target promoters are most robust, backupped by many activators. Second, in contrast to most laboratory strains, the widely used BY4741 strain does not induce Msn2/4 activity when the Ras/cAMP cascade is downregulated.


Assuntos
Proteínas de Choque Térmico/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Fatores de Transcrição de Zíper de Leucina Básica/genética , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico/química , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA