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1.
Nat Commun ; 12(1): 5224, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471130

RESUMO

The replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood. Here, we report the characterization of a chromatin remodeling enzyme-Yta7-entirely distinct from classical SNF2-ATPase family remodelers. Yta7 is a AAA+ -ATPase that assembles into ~1 MDa hexameric complexes capable of segregating histones from DNA. The Yta7 chromatin segregase promotes chromosome replication both in vivo and in vitro. Biochemical reconstitution experiments using purified proteins revealed that the enzymatic activity of Yta7 is regulated by S phase-forms of Cyclin-Dependent Kinase (S-CDK). S-CDK phosphorylation stimulates ATP hydrolysis by Yta7, promoting nucleosome disassembly and chromatin replication. Our results present a mechanism for how cells orchestrate chromatin dynamics in co-ordination with the cell cycle machinery to promote genome duplication during S phase.


Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Replicação do DNA/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Pontos de Checagem do Ciclo Celular , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/genética , DNA/metabolismo , Histonas/metabolismo , Humanos , Fosforilação , Fase S , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição
2.
Nat Commun ; 12(1): 5254, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489436

RESUMO

Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Pdr5 and its homologues drive drug efflux through uncoupled hydrolysis of nucleotides, enabling organisms such as baker's yeast and pathogenic fungi to survive in the presence of chemically diverse antifungal agents. Here, we present the molecular structure of Pdr5 solved with single particle cryo-EM, revealing details of an ATP-driven conformational cycle, which mechanically drives drug translocation through an amphipathic channel, and a clamping switch within a conserved linker loop that acts as a nucleotide sensor. One half of the transporter remains nearly invariant throughout the cycle, while its partner undergoes changes that are transmitted across inter-domain interfaces to support a peristaltic motion of the pumped molecule. The efflux model proposed here rationalises the pleiotropic impact of Pdr5 and opens new avenues for the development of effective antifungal compounds.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Detergentes/química , Farmacorresistência Fúngica/genética , Pleiotropia Genética , Hidrólise , Mutação , Conformação Proteica , Domínios Proteicos , Rodaminas/química , Rodaminas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vanadatos/química , Vanadatos/metabolismo
3.
BMC Genomics ; 22(1): 688, 2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34551706

RESUMO

BACKGROUND: Eukaryotic organisms, like the model yeast S. cerevisiae, have linear chromosomes that facilitate organization and protection of nuclear DNA. A recent work described a stepwise break/repair method that enabled fusion of the 16 chromosomes of S. cerevisiae into a single large chromosome. Construction of this strain resulted in the removal of 30 of 32 telomeres, over 300 kb of subtelomeric DNA, and 107 subtelomeric ORFs. Despite these changes, characterization of the single chromosome strain uncovered modest phenotypes compared to a reference strain. RESULTS: This study further characterized the single chromosome strain and found that it exhibited a longer lag phase, increased doubling time, and lower final biomass concentration compared with a reference strain when grown on YPD. These phenotypes were amplified when ethanol was added to the medium or used as the sole carbon source. RNAseq analysis showed poor induction of genes involved in diauxic shift, ethanol metabolism, and fatty-acid ß-oxidation during growth on ethanol compared to the reference strain. Enzyme-constrained metabolic modeling identified decreased flux through the enzymes that are encoded by these poorly induced genes as a likely cause of diminished biomass accumulation. The diminished growth on ethanol for the single chromosome strain was rescued by nicotinamide, an inhibitor of sirtuin family deacetylases, which have been shown to silence gene expression in heterochromatic regions. CONCLUSIONS: Our results indicate that sirtuin-mediated silencing in the single chromosome strain interferes with growth on non-fermentable carbon sources. We propose that the removal of subtelomeric DNA that would otherwise be bound by sirtuins leads to silencing at other loci in the single chromosome strain. Further, we hypothesize that the poorly induced genes in the single chromosome strain during ethanol growth could be silenced by sirtuins in wildtype S. cerevisiae during growth on glucose.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Tolerância a Medicamentos , Etanol , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Telômero/genética
4.
Methods Enzymol ; 658: 359-377, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517954

RESUMO

A tRNA interacts with numerous proteins throughout its biogenesis and during translation, and a significant portion of these interacting proteins are involved in post-transcriptional modifications. While some of the modifying enzymes use relatively simple recognition elements for substrate recognition, many enzymes selectively modify a specific subset of tRNA species without obvious recognition rules. In this chapter we describe a semi-quantitative pull-down assay to study tRNA substrate specificity of modification enzymes, by using the yeast Saccharomyces cerevisiae m3C32 methyltransferase Trm140 as an example. We also discuss some overall considerations for a successful pull-down experiment, with a focus on practical applications of the dissociation constant KD between the protein and the tRNA and the off-rate.


Assuntos
Proteínas de Saccharomyces cerevisiae , tRNA Metiltransferases , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
5.
Nat Commun ; 12(1): 4918, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389719

RESUMO

Ribosomal RNA genes (rDNA) are highly unstable and susceptible to rearrangement due to their repetitive nature and active transcriptional status. Sequestration of rDNA in the nucleolus suppresses uncontrolled recombination. However, broken repeats must be first released to the nucleoplasm to allow repair by homologous recombination. Nucleolar release of broken rDNA repeats is conserved from yeast to humans, but the underlying molecular mechanisms are currently unknown. Here we show that DNA damage induces phosphorylation of the CLIP-cohibin complex, releasing membrane-tethered rDNA from the nucleolus in Saccharomyces cerevisiae. Downstream of phosphorylation, SUMOylation of CLIP-cohibin is recognized by Ufd1 via its SUMO-interacting motif, which targets the complex for disassembly through the Cdc48/p97 chaperone. Consistent with a conserved mechanism, UFD1L depletion in human cells impairs rDNA release. The dynamic and regulated assembly and disassembly of the rDNA-tethering complex is therefore a key determinant of nucleolar rDNA release and genome integrity.


Assuntos
Nucléolo Celular/genética , Reparo do DNA , DNA Ribossômico/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteína com Valosina/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Nucléolo Celular/metabolismo , Dano ao DNA , DNA Ribossômico/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Técnicas do Sistema de Duplo-Híbrido , Proteína com Valosina/metabolismo
6.
Nucleic Acids Res ; 49(15): 8535-8555, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34358317

RESUMO

Gene deletion and gene expression alteration can lead to growth defects that are amplified or reduced when a second mutation is present in the same cells. We performed 154 genetic interaction mapping (GIM) screens with query mutants related with RNA metabolism and estimated the growth rates of about 700 000 double mutant Saccharomyces cerevisiae strains. The tested targets included the gene deletion collection and 900 strains in which essential genes were affected by mRNA destabilization (DAmP). To analyze the results, we developed RECAP, a strategy that validates genetic interaction profiles by comparison with gene co-citation frequency, and identified links between 1471 genes and 117 biological processes. In addition to these large-scale results, we validated both enhancement and suppression of slow growth measured for specific RNA-related pathways. Thus, negative genetic interactions identified a role for the OCA inositol polyphosphate hydrolase complex in mRNA translation initiation. By analysis of suppressors, we found that Puf4, a Pumilio family RNA binding protein, inhibits ribosomal protein Rpl9 function, by acting on a conserved UGUAcauUA motif located downstream the stop codon of the RPL9B mRNA. Altogether, the results and their analysis should represent a useful resource for discovery of gene function in yeast.


Assuntos
Genes Fúngicos , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/genética , Alelos , Deleção de Genes , Pleiotropia Genética , Fosfatos de Inositol/metabolismo , Iniciação Traducional da Cadeia Peptídica , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia
7.
Int J Mol Sci ; 22(16)2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34445460

RESUMO

Yeast phenotypes associated with the lack of wobble uridine (U34) modifications in tRNA were shown to be modulated by an allelic variation of SSD1, a gene encoding an mRNA-binding protein. We demonstrate that phenotypes caused by the loss of Deg1-dependent tRNA pseudouridylation are similarly affected by SSD1 allelic status. Temperature sensitivity and protein aggregation are elevated in deg1 mutants and further increased in the presence of the ssd1-d allele, which encodes a truncated form of Ssd1. In addition, chronological lifespan is reduced in a deg1 ssd1-d mutant, and the negative genetic interactions of the U34 modifier genes ELP3 and URM1 with DEG1 are aggravated by ssd1-d. A loss of function mutation in SSD1, ELP3, and DEG1 induces pleiotropic and overlapping phenotypes, including sensitivity against target of rapamycin (TOR) inhibitor drug and cell wall stress by calcofluor white. Additivity in ssd1 deg1 double mutant phenotypes suggests independent roles of Ssd1 and tRNA modifications in TOR signaling and cell wall integrity. However, other tRNA modification defects cause growth and drug sensitivity phenotypes, which are not further intensified in tandem with ssd1-d. Thus, we observed a modification-specific rather than general effect of SSD1 status on phenotypic variation in tRNA modification mutants. Our results highlight how the cellular consequences of tRNA modification loss can be influenced by protein targeting specific mRNAs.


Assuntos
Transferases Intramoleculares/deficiência , Processamento Pós-Transcricional do RNA/genética , RNA Fúngico , RNA de Transferência , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Variação Biológica da População , Transferases Intramoleculares/genética , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Int J Mol Sci ; 22(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34445202

RESUMO

The yeast Saccharomyces cerevisiae is one of the most widely used model organisms for investigating various aspects of basic cellular functions that are conserved in human cells. This organism, as well as human cells, can modulate its metabolism in response to specific growth conditions, different environmental changes, and nutrient depletion. This adaptation results in a metabolic reprogramming of specific metabolic pathways. Mitochondrial carriers play a fundamental role in cellular metabolism, connecting mitochondrial with cytosolic reactions. By transporting substrates across the inner membrane of mitochondria, they contribute to many processes that are central to cellular function. The genome of Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family, most of which have been functionally characterized. The aim of this review is to describe the role of the so far identified yeast mitochondrial carriers in cell metabolism, attempting to show the functional connections between substrates transport and specific metabolic pathways, such as oxidative phosphorylation, lipid metabolism, gluconeogenesis, and amino acids synthesis. Analysis of the literature reveals that these proteins transport substrates involved in the same metabolic pathway with a high degree of flexibility and coordination. The understanding of the role of mitochondrial carriers in yeast biology and metabolism could be useful for clarifying unexplored aspects related to the mitochondrial carrier network. Such knowledge will hopefully help in obtaining more insight into the molecular basis of human diseases.


Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico Ativo , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
9.
Int J Mol Sci ; 22(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34445649

RESUMO

Protein aggregation is associated with a growing list of human diseases. A substantial fraction of proteins in eukaryotic proteomes constitutes a proteostasis network-a collection of proteins that work together to maintain properly folded proteins. One of the overarching functions of the proteostasis network is the prevention or reversal of protein aggregation. How proteins aggregate in spite of the anti-aggregation activity of the proteostasis machinery is incompletely understood. Exposed hydrophobic patches can trigger degradation by the ubiquitin-proteasome system, a key branch of the proteostasis network. However, in a recent study, we found that model glycine (G)-rich or glutamine/asparagine (Q/N)-rich prion-like domains differ in their susceptibility to detection and degradation by this system. Here, we expand upon this work by examining whether the features controlling the degradation of our model prion-like domains generalize broadly to G-rich and Q/N-rich domains. Experimentally, native yeast G-rich domains in isolation are sensitive to the degradation-promoting effects of hydrophobic residues, whereas native Q/N-rich domains completely resist these effects and tend to aggregate instead. Bioinformatic analyses indicate that native G-rich domains from yeast and humans tend to avoid degradation-promoting features, suggesting that the proteostasis network may act as a form of selection at the molecular level that constrains the sequence space accessible to G-rich domains. However, the sensitivity or resistance of G-rich and Q/N-rich domains, respectively, was not always preserved in their native protein contexts, highlighting that proteins can evolve other sequence features to overcome the intrinsic sensitivity of some LCDs to degradation.


Assuntos
Agregados Proteicos/fisiologia , Proteoma/metabolismo , Proteostase , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
10.
Nat Commun ; 12(1): 4769, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362905

RESUMO

Beyond its role in mitochondrial bioenergetics, Coenzyme Q (CoQ, ubiquinone) serves as a key membrane-embedded antioxidant throughout the cell. However, how CoQ is mobilized from its site of synthesis on the inner mitochondrial membrane to other sites of action remains a longstanding mystery. Here, using a combination of Saccharomyces cerevisiae genetics, biochemical fractionation, and lipid profiling, we identify two highly conserved but poorly characterized mitochondrial proteins, Ypl109c (Cqd1) and Ylr253w (Cqd2), that reciprocally affect this process. Loss of Cqd1 skews cellular CoQ distribution away from mitochondria, resulting in markedly enhanced resistance to oxidative stress caused by exogenous polyunsaturated fatty acids, whereas loss of Cqd2 promotes the opposite effects. The activities of both proteins rely on their atypical kinase/ATPase domains, which they share with Coq8-an essential auxiliary protein for CoQ biosynthesis. Overall, our results reveal protein machinery central to CoQ trafficking in yeast and lend insights into the broader interplay between mitochondria and the rest of the cell.


Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo , Antioxidantes/metabolismo , Lipídeos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Estresse Oxidativo , Fosfotransferases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
11.
Nat Commun ; 12(1): 4951, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400637

RESUMO

The polyadenosine tail (poly[A]-tail) is a universal modification of eukaryotic messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs). In budding yeast, Pap1-synthesized mRNA poly(A) tails enhance export and translation, whereas Trf4/5-mediated polyadenylation of ncRNAs facilitates degradation by the exosome. Using direct RNA sequencing, we decipher the extent of poly(A) tail dynamics in yeast defective in all relevant exonucleases, deadenylases, and poly(A) polymerases. Predominantly ncRNA poly(A) tails are 20-60 adenosines long. Poly(A) tails of newly transcribed mRNAs are 50 adenosine long on average, with an upper limit of 200. Exonucleolysis by Trf5-assisted nuclear exosome and cytoplasmic deadenylases trim the tails to 40 adenosines on average. Surprisingly, PAN2/3 and CCR4-NOT deadenylase complexes have a large pool of non-overlapping substrates mainly defined by expression level. Finally, we demonstrate that mRNA poly(A) tail length strongly responds to growth conditions, such as heat and nutrient deprivation.


Assuntos
Poli A/metabolismo , Polinucleotídeo Adenililtransferase/metabolismo , RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Exossomos/metabolismo , Poliadenilação , Polinucleotídeo Adenililtransferase/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
Nat Commun ; 12(1): 4696, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34349113

RESUMO

Productive ribosomal RNA (rRNA) compaction during ribosome assembly necessitates establishing correct tertiary contacts between distant secondary structure elements. Here, we quantify the response of the yeast proteome to low temperature (LT), a condition where aberrant mis-paired RNA folding intermediates accumulate. We show that, at LT, yeast cells globally boost production of their ribosome assembly machinery. We find that the LT-induced assembly factor, Puf6, binds to the nascent catalytic RNA-rich subunit interface within the 60S pre-ribosome, at a site that eventually loads the nuclear export apparatus. Ensemble Förster resonance energy transfer studies show that Puf6 mimics the role of Mg2+ to usher a unique long-range tertiary contact to compact rRNA. At LT, puf6 mutants accumulate 60S pre-ribosomes in the nucleus, thus unveiling Puf6-mediated rRNA compaction as a critical temperature-regulated rescue mechanism that counters rRNA misfolding to prime export competence.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a RNA/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Temperatura Baixa , GTP Fosfo-Hidrolases/metabolismo , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteoma/metabolismo , Dobramento de RNA , Precursores de RNA/química , Precursores de RNA/metabolismo , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Subunidades Ribossômicas Maiores de Eucariotos/química , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
13.
J Cell Sci ; 134(2)2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-34432037

RESUMO

Eukaryotic cells adapt their metabolism to the extracellular environment. Downregulation of surface cargo proteins in response to nutrient stress reduces the burden of anabolic processes whilst elevating catabolic production in the lysosome. We show that glucose starvation in yeast triggers a transcriptional response that increases internalisation from the plasma membrane. Nuclear export of the Mig1 transcriptional repressor in response to glucose starvation increases levels of the Yap1801 and Yap1802 clathrin adaptors, which is sufficient to increase cargo internalisation. Beyond this, we show that glucose starvation results in Mig1-independent transcriptional upregulation of various eisosomal factors. These factors serve to sequester a portion of nutrient transporters at existing eisosomes, through the presence of Ygr130c and biochemical and biophysical changes in Pil1, allowing cells to persist throughout the starvation period and maximise nutrient uptake upon return to replete conditions. This provides a physiological benefit for cells to rapidly recover from glucose starvation. Collectively, this remodelling of the surface protein landscape during glucose starvation calibrates metabolism to available nutrients. This article has an associated First Person interview with the first author of the paper.


Assuntos
Glucose , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Membrana Celular , Fosfoproteínas , Proteínas Repressoras , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
14.
Nat Commun ; 12(1): 4451, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294712

RESUMO

Identifying how R-loops are generated is crucial to know how transcription compromises genome integrity. We show by genome-wide analysis of conditional yeast mutants that the THO transcription complex, prevents R-loop formation in G1 and S-phase, whereas the Sen1 DNA-RNA helicase prevents them only in S-phase. Interestingly, damage accumulates asymmetrically downstream of the replication fork in sen1 cells but symmetrically in the hpr1 THO mutant. Our results indicate that: R-loops form co-transcriptionally independently of DNA replication; that THO is a general and cell-cycle independent safeguard against R-loops, and that Sen1, in contrast to previously believed, is an S-phase-specific R-loop resolvase. These conclusions have important implications for the mechanism of R-loop formation and the role of other factors reported to affect on R-loop homeostasis.


Assuntos
DNA Fúngico/química , Estruturas R-Loop , RNA Fúngico/química , Ciclo Celular/genética , Ciclo Celular/fisiologia , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Genes Fúngicos , Instabilidade Genômica , Modelos Biológicos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estruturas R-Loop/genética , Estruturas R-Loop/fisiologia , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
Adv Exp Med Biol ; 1208: 43-53, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34260020

RESUMO

Phagophore closure is a critical step during macroautophagy. However, the proteins and mechanisms to regulate this step have been elusive for a long time. In 2017, Rab5 was affirmed to play a role in phagophore closure in yeast. Furthermore, in mammalian cells, ESCRT III was reported to have roles in phagophore closure and mitophagosome closure in vivo in 2018 and 2019, respectively. The role of ESCRT in phagophore closure was confirmed in yeast, both in vivo and in vitro, in 2019. Most importantly, the latter paper found that Atg17 recruited the ESCRT III subunit Snf7 to the phagophore to close it under the control of Rab5. To determine the closure characteristics of autophagosome-like membrane structures in ESCRT mutants, a traditional protease protection assay with immunoblotting was used, accompanied by new techniques that were developed, including immunofluorescence assays, autophagosome completion assays, and the optogenetic closure assay. This study delivered our current understanding of phagophore closure and provided more reference methods to detect membrane closure.


Assuntos
Autofagossomos , Proteínas de Saccharomyces cerevisiae , Animais , Autofagia , Complexos Endossomais de Distribuição Requeridos para Transporte , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
16.
Enzyme Microb Technol ; 149: 109848, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34311885

RESUMO

The signal peptide sequence is known to increase transport efficiency to organelles in eukaryotic cells. In this study, we focus on the signal peptide of the vacuolar protein for vacuolar targeting. The signal peptide sequence QRPL of carboxypeptidase Y (CPY) was inserted inside the interest protein that does not locate in the vacuole for vacuolar targeting. We constructed recombinant strains MBTL-Q-DJ1 and MBTL-Q-DJ2 containing QRPL and green florescent protein (GFP) or aldehyde dehydrogenase 6 (ALD6), respectively. The protein location was then confirmed by confocal microscopy. Fascinatingly, the green fluorescent protein that contains QRPL inside the sequence could be expressed faster than its natural form (within 1 h after induction). Also, the aldehyde removal activity of ALD6 protein in the recombinant yeast was then analyzed by measuring the luminescent intensity in Vibrio fischeri. We confirmed that MBTL-Q-DJ2 containing ALD6 protein has the aldehydes-reducing ability, and in particular, the highest efficiency showed at 500 µg/µL of vacuolar enzyme. In summary, the signal peptide QRPL could be used not only to transport proteins accurately to vacuole but also to improve the protein activity and shorten the induction time.


Assuntos
Proteínas de Saccharomyces cerevisiae , Vacúolos , Catepsina A/genética , Sinais Direcionadores de Proteínas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
17.
Appl Microbiol Biotechnol ; 105(14-15): 5895-5904, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34272577

RESUMO

The expression of functional proteins on the cell surface using glycosylphosphatidylinositol (GPI)-anchoring technology is a promising approach for constructing yeast cells with special functions. The functionality of surface-engineered yeast strains strongly depends on the amount of functional proteins displayed on their cell surface. On the other hand, since the yeast cell wall space is finite, heterologous protein carrying capacity of the cell wall is limited. Here, we report the effect of CCW12 and CCW14 knockout, which encode major nonenzymatic GPI-anchored cell wall proteins (GPI-CWPs) involved in the cell wall organization, on the heterologous protein carrying capacity of yeast cell wall. Aspergillus aculeatus ß-glucosidase (BGL) was used as a reporter to evaluate the protein carrying capacity in Saccharomyces cerevisiae. No significant difference in the amount of cell wall-associated BGL and cell-surface BGL activity was observed between CCW12 and CCW14 knockout strains and their control strain. In contrast, in the CCW12 and CCW14 co-knockout strains, the amount of cell wall-associated BGL and its activity were approximately 1.4-fold higher than those of the control strain and CCW12 or CCW14 knockout strains. Electron microscopic observation revealed that the total cell wall thickness of the CCW12 and CCW14 co-knockout strains was increased compared to the parental strain, suggesting a potential increase in heterologous protein carrying capacity of the cell wall. These results indicate that the CCW12 and CCW14 co-knockout strains are a promising host for the construction of highly functional recombinant yeast strains using cell-surface display technology. KEY POINTS: • CCW12 and/or CCW14 of a BGL-displaying S. cerevisiae strain were knocked out. • CCW12 and CCW14 co-disruption improved the display efficiency of BGL. • The thickness of the yeast cell wall was increased upon CCW12 and CCW14 knockout.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Aspergillus , Parede Celular , Glicosilfosfatidilinositóis , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
18.
Appl Microbiol Biotechnol ; 105(14-15): 5809-5819, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34283270

RESUMO

As a natural phenolic acid product of plant source, caffeic acid displays diverse biological activities and acts as an important precursor for the synthesis of other valuable compounds. Limitations in chemical synthesis or plant extraction of caffeic acid trigger interest in its microbial biosynthesis. Recently, Saccharomyces cerevisiae has been reported for the biosynthesis of caffeic acid via episomal plasmid-mediated expression of pathway genes. However, the production was far from satisfactory and even relied on the addition of precursor. In this study, we first established a controllable and stable caffeic acid pathway by employing a modified GAL regulatory system to control the genome-integrated pathway genes in S. cerevisiae and realized biosynthesis of 222.7 mg/L caffeic acid. Combinatorial engineering strategies including eliminating the tyrosine-induced feedback inhibition, deleting genes involved in competing pathways, and overexpressing rate-limiting enzymes led to about 2.6-fold improvement in the caffeic acid production, reaching up to 569.0 mg/L in shake-flask cultures. To our knowledge, this is the highest ever reported titer of caffeic acid synthesized by engineered yeast. This work showed the prospect for microbial biosynthesis of caffeic acid and laid the foundation for constructing biosynthetic pathways of its derived metabolites. KEY POINTS: Genomic integration of ORgTAL, OHpaB, and HpaC for caffeic acid production in yeast. Feedback inhibition elimination and Aro10 deletion improved caffeic acid production. The highest ever reported titer (569.0 mg/L) of caffeic acid synthesized by yeast.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ácidos Cafeicos , Engenharia Metabólica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
19.
Nat Commun ; 12(1): 4202, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244507

RESUMO

Biochemical reactions typically depend on the concentrations of the molecules involved, and cell survival therefore critically depends on the concentration of proteins. To maintain constant protein concentrations during cell growth, global mRNA and protein synthesis rates are tightly linked to cell volume. While such regulation is appropriate for most proteins, certain cellular structures do not scale with cell volume. The most striking example of this is the genomic DNA, which doubles during the cell cycle and increases with ploidy, but is independent of cell volume. Here, we show that the amount of histone proteins is coupled to the DNA content, even though mRNA and protein synthesis globally increase with cell volume. As a consequence, and in contrast to the global trend, histone concentrations decrease with cell volume but increase with ploidy. We find that this distinct coordination of histone homeostasis and genome content is already achieved at the transcript level, and is an intrinsic property of histone promoters that does not require direct feedback mechanisms. Mathematical modeling and histone promoter truncations reveal a simple and generalizable mechanism to control the cell volume- and ploidy-dependence of a given gene through the balance of the initiation and elongation rates.


Assuntos
Histonas/biossíntese , Modelos Genéticos , Biossíntese de Proteínas/genética , RNA Mensageiro/biossíntese , Transcrição Genética , DNA Fúngico/genética , Genoma Fúngico , Histonas/genética , Ploidias , Regiões Promotoras Genéticas/genética , RNA Fúngico/biossíntese , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética
20.
Nat Commun ; 12(1): 4044, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193872

RESUMO

What determines the rate (µ) and molecular spectrum of mutation is a fundamental question. The prevailing hypothesis asserts that natural selection against deleterious mutations has pushed µ to the minimum achievable in the presence of genetic drift, or the drift barrier. Here we show that, contrasting this hypothesis, µ substantially exceeds the drift barrier in diverse organisms. Random mutation accumulation (MA) in yeast frequently reduces µ, and deleting the newly discovered mutator gene PSP2 nearly halves µ. These results, along with a comparison between the MA and natural yeast strains, demonstrate that µ is maintained above the drift barrier by stabilizing selection. Similar comparisons show that the mutation spectrum such as the universal AT mutational bias is not intrinsic but has been selectively preserved. These findings blur the separation of mutation from selection as distinct evolutionary forces but open the door to alleviating mutagenesis in various organisms by genome editing.


Assuntos
Modelos Genéticos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Deriva Genética , Acúmulo de Mutações , Taxa de Mutação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Seleção Genética , Sequenciamento Completo do Genoma/métodos
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