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1.
Elife ; 102021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34545808

RESUMO

In fluctuating environments, switching between different growth strategies, such as those affecting cell size and proliferation, can be advantageous to an organism. Trade-offs arise, however. Mechanisms that aberrantly increase cell size or proliferation-such as mutations or chemicals that interfere with growth regulatory pathways-can also shorten lifespan. Here we report a natural example of how the interplay between growth and lifespan can be epigenetically controlled. We find that a highly conserved RNA-modifying enzyme, the pseudouridine synthase Pus4/TruB, can act as a prion, endowing yeast with greater proliferation rates at the cost of a shortened lifespan. Cells harboring the prion grow larger and exhibit altered protein synthesis. This epigenetic state, [BIG+] (better in growth), allows cells to heritably yet reversibly alter their translational program, leading to the differential synthesis of dozens of proteins, including many that regulate proliferation and aging. Our data reveal a new role for prion-based control of an RNA-modifying enzyme in driving heritable epigenetic states that transform cell growth and survival.


Assuntos
Proliferação de Células , Transferases Intramoleculares/metabolismo , Meiose , Proteínas Priônicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Crescimento Celular , Epigênese Genética , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Transferases Intramoleculares/genética , Longevidade , Proteínas Priônicas/genética , Biossíntese de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Tempo
2.
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
3.
FEBS Lett ; 595(18): 2383-2394, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34358326

RESUMO

Maintenance of the proteome (proteostasis) is essential for cellular homeostasis and prevents cytotoxic stress responses that arise from protein misfolding. However, little is known about how different types of misfolded proteins impact homeostasis, especially when protein degradation pathways are compromised. We examined the effects of misfolded protein expression on yeast growth by characterizing a suite of substrates possessing the same aggregation-prone domain but engaging different quality control pathways. We discovered that treatment with a proteasome inhibitor was more toxic in yeast expressing misfolded membrane proteins, and this growth defect was mirrored in yeast lacking a proteasome-specific transcription factor, Rpn4p. These results highlight weaknesses in the proteostasis network's ability to handle the stress arising from an accumulation of misfolded membrane proteins.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/classificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Processos de Crescimento Celular/efeitos dos fármacos , Citoplasma/metabolismo , Proteínas de Ligação a DNA/deficiência , Degradação Associada com o Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Nucleotídeos/metabolismo , Inibidores de Proteassoma/farmacologia , Ligação Proteica , Domínios Proteicos , Proteólise , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/deficiência
4.
FEBS Lett ; 595(17): 2208-2220, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34293820

RESUMO

Previous studies have suggested that phosphorylation of translation elongation factor 1A (eEF1A) can alter its function, and large-scale phospho-proteomic analyses in Saccharomyces cerevisiae have identified 14 eEF1A residues phosphorylated under various conditions. Here, a series of eEF1A mutations at these proposed sites were created and the effects on eEF1A activity were analyzed. The eEF1A-S53D and eEF1A-T430D phosphomimetic mutant strains were inviable, while corresponding alanine mutants survived but displayed defects in growth and protein synthesis. The activity of an eEF1A-S289D mutant was significantly reduced in the absence of the guanine nucleotide exchange factor eEF1Bα and could be restored by an exchange-deficient form of the protein, suggesting that eEF1Bα promotes eEF1A activity by a mechanism other than nucleotide exchange. Our data show that several of the phosphorylation sites identified by high-throughput analysis are critical for eEF1A function.


Assuntos
Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise Mutacional de DNA , Fosforilação , Biossíntese de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento
5.
Int J Mol Sci ; 22(12)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201004

RESUMO

Cadmium is a carcinogen that can induce ER stress, DNA damage, oxidative stress and cell death. The yeast mitogen-activated protein kinase (MAPK) signalling pathways paly crucial roles in response to various stresses. Here, we demonstrate that the unfolded protein response (UPR) pathway, the high osmolarity glycerol (HOG) pathway and the cell wall integrity (CWI) pathway are all essential for yeast cells to defend against the cadmium-induced toxicity, including the elevated ROS and cell death levels induced by cadmium. We show that the UPR pathway is required for the cadmium-induced phosphorylation of HOG_MAPK Hog1 but not for CWI_MAPK Slt2, while Slt2 but not Hog1 is required for the activation of the UPR pathway through the transcription factors of Swi6 and Rlm1. Moreover, deletion of HAC1 and IRE1 could promote the nuclear accumulation of Hog1, and increase the cytosolic and bud neck localisation of Slt2, indicating crucial roles of Hog1 and Slt2 in regulating the cellular process in the absence of UPR pathway. Altogether, our findings highlight the significance of these two MAPK pathways of HOG and CWI and their interrelationship with the UPR pathway in responding to cadmium-induced toxicity in budding yeast.


Assuntos
Cádmio/toxicidade , Parede Celular/química , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glicerol/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Parede Celular/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Concentração Osmolar , Fosforilação , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transdução de Sinais
6.
Int J Mol Sci ; 22(12)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201352

RESUMO

The VPS13 family of proteins have emerged as key players in intracellular lipid transport and human health. Humans have four different VPS13 orthologs, the dysfunction of which leads to different diseases. Yeast has a single VPS13 gene, which encodes a protein that localizes to multiple different membrane contact sites. The yeast vps13Δ mutant is pleiotropic, exhibiting defects in sporulation, protein trafficking, endoplasmic reticulum (ER)-phagy and mitochondrial function. Non-null alleles resulting from missense mutations can be useful reagents for understanding the multiple functions of a gene. The exceptionally large size of Vps13 makes the identification of key residues challenging. As a means to identify critical residues in yeast Vps13, amino acid substitution mutations from VPS13A, B, C and D, associated with human disease, were introduced at the cognate positions of yeast VPS13, some of which created separation-of-function alleles. Phenotypic analyses of these mutants have revealed that the promotion of ER-phagy is a fourth, genetically separable role of VPS13 and provide evidence that co-adaptors at the endosome mediate the activity of VPS13 in vacuolar sorting.


Assuntos
Mitocôndrias/metabolismo , Mutação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética
7.
Int J Mol Sci ; 22(14)2021 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-34299038

RESUMO

Ribosomal RNA is a major component of the ribosome. This RNA plays a crucial role in ribosome functioning by ensuring the formation of the peptide bond between amino acids and the accurate decoding of the genetic code. The rRNA carries many chemical modifications that participate in its maturation, the formation of the ribosome and its functioning. In this review, we present the different modifications and how they are deposited on the rRNA. We also describe the most recent results showing that the modified positions are not 100% modified, which creates a heterogeneous population of ribosomes. This gave rise to the concept of specialized ribosomes that we discuss. The knowledge accumulated in the yeast Saccharomyces cerevisiae is very helpful to better understand the role of rRNA modifications in humans, especially in ribosomopathies.


Assuntos
Modelos Biológicos , Processamento Pós-Transcricional do RNA , RNA Fúngico/genética , RNA Ribossômico/genética , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Humanos , Ribossomos/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
8.
Food Microbiol ; 99: 103806, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34119099

RESUMO

The increasing interest in novel beer productions focused on non-Saccharomyces yeasts in order to pursue their potential in generating groundbreaking sensory profiles. Traditional fermented beverages represent an important source of yeast strains which could express interesting features during brewing. A total of 404 yeasts were isolated from fermented honey by-products and identified as Saccharomyces cerevisiae, Wickerhamomyces anomalus, Zygosaccharomyces bailii, Zygosaccharomyces rouxii and Hanseniaspora uvarum. Five H. uvarum strains were screened for their brewing capability. Interestingly, Hanseniaspora uvarum strains showed growth in presence of ethanol and hop and a more rapid growth than the control strain S. cerevisiae US-05. Even though all strains showed a very low fermentation power, their concentrations ranged between 7 and 8 Log cycles during fermentation. The statistical analyses showed significant differences among the strains and underlined the ability of YGA2 and YGA34 to grow rapidly in presence of ethanol and hop. The strain YGA34 showed the best technological properties and was selected for beer production. Its presence in mixed- and sequential-culture fermentations with US-05 did not influence attenuation and ethanol concentration but had a significant impact on glycerol and acetic acid concentrations, with a higher sensory complexity and intensity, representing promising co-starters during craft beer production.


Assuntos
Cerveja/microbiologia , Hanseniaspora/metabolismo , Mel/microbiologia , Ácido Acético/análise , Ácido Acético/metabolismo , Cerveja/análise , Etanol/metabolismo , Fermentação , Microbiologia de Alimentos , Hanseniaspora/crescimento & desenvolvimento , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Resíduos/análise , Leveduras/crescimento & desenvolvimento , Leveduras/metabolismo
9.
Appl Environ Microbiol ; 87(16): e0058821, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34105981

RESUMO

High ethanol levels can severely inhibit the growth of yeast cells and fermentation productivity. The ethanologenic yeast Saccharomyces cerevisiae activates several well-defined cellular mechanisms of ethanol stress response (ESR); however, the involved regulatory control remains to be characterized. Here, we report a new transcription factor of ethanol stress adaptation called Znf1. It plays a central role in ESR by activating genes for glycerol and fatty acid production (GUP1, GPP1, GPP2, GPD1, GAT1, and OLE1) to preserve plasma membrane integrity. Importantly, Znf1 also activates genes implicated in cell wall biosynthesis (FKS1, SED1, and SMI1) and in the unfolded protein response (HSP30, HSP104, KAR1, and LHS1) to protect cells from proteotoxic stress. The znf1Δ strain displays increased sensitivity to ethanol, the endoplasmic reticulum (ER) stressor ß-mercaptoethanol, and the cell wall-perturbing agent calcofluor white. To compensate for a defective cell wall, the strain lacking ZNF1 or its target SMI1 displays increased glycerol levels of 19.6% and 27.7%, respectively. Znf1 collectively regulates an intricate network of target genes essential for growth, protein refolding, and production of key metabolites. Overexpression of ZNF1 not only confers tolerance to high ethanol levels but also increases ethanol production by 4.6% (8.43 g/liter) or 2.8% (75.78 g/liter) when 2% or 20% (wt/vol) glucose, respectively, is used as a substrate, compared to that of the wild-type strain. The mutually stress-responsive transcription factors Msn2/4, Hsf1, and Yap1 are associated with some promoters of Znf1's target genes to promote ethanol stress tolerance. In conclusion, this work implicates the novel regulator Znf1 in coordinating expression of ESR genes and illuminates the unifying transcriptional reprogramming during alcoholic fermentation. IMPORTANCE The yeast S. cerevisiae is a major microbe that is widely used in food and nonfood industries. However, accumulation of ethanol has a negative effect on its growth and limits ethanol production. The Znf1 transcription factor has been implicated as a key regulator of glycolysis and gluconeogenesis in the utilization of different carbon sources, including glucose, the most abundant sugar on earth, and nonfermentable substrates. Here, the role of Znf1 in ethanol stress response is defined. Znf1 actively reprograms expression of genes linked to the unfolded protein response (UPR), heat shock response, glycerol and carbohydrate metabolism, and biosynthesis of cell membrane and cell wall components. A complex interplay among transcription factors of ESR indicates transcriptional fine-tuning as the main mechanism of stress adaptation, and Znf1 plays a major regulatory role in the coordination. Understanding the adaptive ethanol stress mechanism is crucial to engineering robust yeast strains for enhanced stress tolerance or increased ethanol production.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Etanol/metabolismo , Glicerol/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Vias Biossintéticas , Proteínas de Ligação a DNA/genética , Fermentação , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
10.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068231

RESUMO

Stress granules (SGs) are membrane-less assemblies arising upon various stresses in eukaryotic cells. They sequester mRNAs and proteins from stressful conditions and modulate gene expression to enable cells to resume translation and growth after stress relief. SGs containing the translation initiation factor eIF3a/Rpg1 arise in yeast cells upon robust heat shock (HS) at 46 °C only. We demonstrate that the destabilization of Rpg1 within the PCI domain in the Rpg1-3 variant leads to SGs assembly already at moderate HS at 42 °C. These are bona fide SGs arising upon translation arrest containing mRNAs, which are components of the translation machinery, and associating with P-bodies. HS SGs associate with endoplasmatic reticulum and mitochondria and their contact sites ERMES. Although Rpg1-3-labeled SGs arise at a lower temperature, their disassembly is delayed after HS at 46 °C. Remarkably, the delayed disassembly of HS SGs after the robust HS is reversed by TDP-43, which is a human protein connected with amyotrophic lateral sclerosis. TDP-43 colocalizes with HS SGs in yeast cells and facilitates cell regrowth after the stress relief. Based on our results, we propose yeast HS SGs labeled by Rpg1 and its variants as a novel model system to study functions of TDP-43 in stress granules disassembly.


Assuntos
Grânulos Citoplasmáticos/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fator de Iniciação 3 em Eucariotos/química , Resposta ao Choque Térmico , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Humanos , Mitocôndrias/metabolismo , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
11.
Nucleic Acids Res ; 49(12): 7053-7074, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34125911

RESUMO

Eukaryotic ribosome biogenesis is an elaborate process during which ribosomal proteins assemble with the pre-rRNA while it is being processed and folded. Hundreds of assembly factors (AF) are required and transiently recruited to assist the sequential remodeling events. One of the most intricate ones is the stepwise removal of the internal transcribed spacer 2 (ITS2), between the 5.8S and 25S rRNAs, that constitutes together with five AFs the pre-60S 'foot'. In the transition from nucleolus to nucleoplasm, Nop53 replaces Erb1 at the basis of the foot and recruits the RNA exosome for the ITS2 cleavage and foot disassembly. Here we comprehensively analyze the impact of Nop53 recruitment on the pre-60S compositional changes. We show that depletion of Nop53, different from nop53 mutants lacking the exosome-interacting motif, not only causes retention of the unprocessed foot in late pre-60S intermediates but also affects the transition from nucleolar state E particle to subsequent nuclear stages. Additionally, we reveal that Nop53 depletion causes the impairment of late maturation events such as Yvh1 recruitment. In light of recently described pre-60S cryo-EM structures, our results provide biochemical evidence for the structural role of Nop53 rearranging and stabilizing the foot interface to assist the Nog2 particle formation.


Assuntos
Proteínas Nucleares/fisiologia , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Fosfatases de Especificidade Dupla/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Biogênese de Organelas , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
FEBS Lett ; 595(14): 1886-1901, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34096057

RESUMO

Eukaryotes harbour a conserved signalling pathway, called General Amino Acid Control (GAAC) in Saccharomyces cerevisiae, for overcoming amino acid starvation. Upon starvation, the protein kinase Gcn2, which phosphorylates the eukaryotic translation initiation factor eIF2α, becomes stimulated to trigger the GAAC response. Genetic studies suggest that Yih1, which is the yeast homolog of mammalian IMPACT and which binds monomeric actin, inhibits Gcn2 when released from actin. Here, we found that D56A substitution in actin (the act1-9 allele) leads to reduced eIF2α phosphorylation, suggesting that the Asp56 residue is required for full Gcn2 activation. In the act1-9 mutant, Yih1 overexpression further enhanced the sensitivity to amino acid starvation-inducing drugs and further impaired eIF2α phosphorylation, suggesting that Gcn2 inhibition was mediated via Yih1. The D56A substitution may impair the actin-Yih1 interaction, directly or indirectly, thereby increasing the amount of Yih1 available to inhibit Gcn2.


Assuntos
Actinas/genética , Substituição de Aminoácidos , Ácido Aspártico/química , Fator de Iniciação 2 em Eucariotos/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Actinas/química , Actinas/metabolismo , Alanina/química , Alanina/metabolismo , Alelos , Ácido Aspártico/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Meios de Cultura/química , Meios de Cultura/farmacologia , Inibidores Enzimáticos/farmacologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Mutação , Fosforilação , Ligação Proteica , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Compostos de Sulfonilureia/farmacologia
13.
Anal Biochem ; 625: 114216, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-33933444

RESUMO

The counting of microorganisms is essential in the area of microbiology, especially in the preparation of inoculum. The main methods for obtaining inoculum are McFarland standard, Neubauer chamber, and plate count. However, the visual comparison is subjective while the counting in the chamber and the plating are technically time-consuming. For this reason, our article aims to correlate the absorbance of the spectrophotometer in the visible ultraviolet region (UV-Vis) with the cell counting in the Neubauer chamber. This study used suspensions of Candida spp. measured at three wavelengths (530, 600, and 700 nm) and counting in a Neubauer chamber. In the next step, curves were adjusted with different polynomials using absorbances and counts. The two best polynomial curve fittings were the Saturation Growth Rate (SGR) and Morgan-Mercer-Flodin (MMF). Therefore, the polynomials were linearized and a direct correlation between absorbance and the number of cells was made. The proposed method proved to be more accurate (5 ± 0.5 × 106) than the comparison with the McFarland turbidity (1-5 x 106) and more practical than plate counting. Predicting the number of cells by UV-Vis is an alternative that reduces the uncertainty of the cell count interval for inoculum preparation.


Assuntos
Saccharomyces cerevisiae/crescimento & desenvolvimento , Espectrofotometria Ultravioleta/métodos , Contagem de Colônia Microbiana
14.
Elife ; 102021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34042048

RESUMO

Quiescence is a reversible G0 state essential for differentiation, regeneration, stem-cell renewal, and immune cell activation. Necessary for long-term survival, quiescent chromatin is compact, hypoacetylated, and transcriptionally inactive. How transcription activates upon cell-cycle re-entry is undefined. Here we report robust, widespread transcription within the first minutes of quiescence exit. During quiescence, the chromatin-remodeling enzyme RSC was already bound to the genes induced upon quiescence exit. RSC depletion caused severe quiescence exit defects: a global decrease in RNA polymerase II (Pol II) loading, Pol II accumulation at transcription start sites, initiation from ectopic upstream loci, and aberrant antisense transcription. These phenomena were due to a combination of highly robust Pol II transcription and severe chromatin defects in the promoter regions and gene bodies. Together, these results uncovered multiple mechanisms by which RSC facilitates initiation and maintenance of large-scale, rapid gene expression despite a globally repressive chromatin state.


Assuntos
Ciclo Celular , Senescência Celular , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Nucleossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transcrição Genética , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismo
15.
Appl Biochem Biotechnol ; 193(9): 2872-2892, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33937964

RESUMO

Microorganisms can produce a wide range of bio-based chemicals that can be used in various industrial applications as molecules of interest. In the present work, an analysis of the power production by pure culture, co-culture, and sequential culture was performed. In this study, both the mono-culture and the co-culture strategies of Actinobacillus succinogenes with Saccharomyces cerevisiae as carbon sources to produce succinic acid using glucose and fructose were examined. The cultures were performed in batch mode and a great attention was paid to the co-culture system to improve the biosynthetic pathway between A. succinogenes and S. cerevisiae by combining these two strains in a single fermentation process. Under microaerobic and anaerobic conditions, the process was characterized in terms of sugars concentration, cell density, metabolites, yield (mol-C products/ mol-C sugars), the temperature conditions for productivity, and pH. The results showed that the process could consume glucose and fructose and could adapt to different concentrations of the two sugars more quickly than by a single organism and the best results were obtained in a sequential co-culture recording 0.27 mol L-1 of succinic acid concentration and a volumetric productivity of 0.3 g L-1 h-1. Under the investigated operating conditions, the combination of these two strains in a single reactor produced a significant amount of succinic acid (0.70 mol-C SA/mol-C substrates). A simultaneous and sequential co-culture strategy can be a powerful new approach in the field of bio-based chemical production.


Assuntos
Actinobacillus/crescimento & desenvolvimento , Saccharomyces cerevisiae/crescimento & desenvolvimento , Ácido Succínico/metabolismo , Técnicas de Cocultura
16.
FASEB J ; 35(6): e21538, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33956347

RESUMO

Two chaperones, Atp23p and Atp10p, were previously shown to regulate the assembly of yeast mitochondrial ATP synthase, and extra expression of ATP23 was found to partially rescue an atp10 deletion mutant, by an unknown mechanism. Here, we identified that the residues 112-115 (LRDK) of Atp23p were required for its function in assisting assembly of the synthase, and demonstrated both functions of Atp23p, processing subunit 6 precursor and assisting assembly of the synthase, were required for the partial rescue of atp10 deletion mutant. By chasing labeling with isotope 35 S-methionine, we found the stability of subunit 6 of the synthase increased in atp10 null strain upon overexpression of ATP23. Further co-immunoprecipitation (Co-IP) and blue native PAGE experiments showed that Atp23p and Atp10p were physically associated with each other in wild type. Moreover, we revealed the expression level of Atp23p increased in atp10 null mutant compared with the wild type. Furthermore, we found that, after 72 hours growth, atp10 null mutant showed leaky growth on respiratory substrates, presence of low level of subunit 6 and partial recovery of oligomycin sensitivity of mitochondrial ATPase activity. Further characterization revealed the expression of Atp23p increased after 24 hours growth in the mutant. These results indicated, in atp10 null mutant, ATP10 deficiency could be partially complemented with increased expression of Atp23p by stabilizing some subunit 6 of the synthase. Taken together, this study revealed the two chaperones Atp23p and Atp10p coordinated to regulate the assembly of mitochondrial ATP synthase, which advanced our understanding of mechanism of assembly of yeast mitochondrial ATP synthase.


Assuntos
Metaloproteases/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Chaperonas Moleculares/metabolismo , Mutação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Metaloproteases/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Chaperonas Moleculares/genética , Mutagênese Sítio-Dirigida , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência
17.
FASEB J ; 35(6): e21615, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33978245

RESUMO

Protein sorting at the trans-Golgi network (TGN) usually requires the assistance of cargo adaptors. However, it remains to be examined how the same complex can mediate both the export and retention of different proteins or how sorting complexes interact among themselves. In Saccharomyces cerevisiae, the exomer complex is involved in the polarized transport of some proteins from the TGN to the plasma membrane (PM). Intriguingly, exomer and its cargos also show a sort of functional relationship with TGN clathrin adaptors that is still unsolved. Here, using a wide range of techniques, including time-lapse and BIFC microscopy, we describe new molecular implications of the exomer complex in protein sorting and address its different layers of functional interaction with clathrin adaptor complexes. Exomer mutants show impaired amino acid uptake because it facilitates not only the polarized delivery of amino acid permeases to the PM but also participates in their endosomal traffic. We propose a model for exomer where it modulates the recruitment of TGN clathrin adaptors directly or indirectly through the Arf1 function. Moreover, we describe an in vivo competitive relationship between the exomer and AP-1 complexes for the model cargo Chs3. These results highlight a broad role for exomer in regulating protein sorting at the TGN that is complementary to its role as cargo adaptor and present a model to understand the complexity of TGN protein sorting.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Quitina Sintase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Rede trans-Golgi/metabolismo , Membrana Celular/metabolismo , Endossomos/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento
18.
Methods Mol Biol ; 2228: 253-270, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950496

RESUMO

Stable isotope labeling by amino acids in cell culture (SILAC) combined with high-resolution mass spectrometry is a quantitative strategy for the comparative analysis of (sub)proteomes. It is based on the metabolic incorporation of stable isotope-coded amino acids during growth of cells or organisms. Here, complete labeling of proteins with the amino acid(s) selected for incorporation needs to be guaranteed to enable accurate quantification on a proteomic scale. Wild-type strains of baker's yeast (Saccharomyces cerevisiae ), which is a widely accepted and well-studied eukaryotic model organism, are generally able to synthesize all amino acids on their own (i.e., prototrophic). To render them amenable to SILAC, auxotrophies are introduced by genetic manipulations. We addressed this limitation by developing a generic strategy for complete "native" labeling of prototrophic S. cerevisiae with isotope-coded arginine and lysine, referred to as "2nSILAC". It allows for directly using and screening several genome-wide yeast mutant collections that are easily accessible to the scientific community for functional proteomic studies but are based on prototrophic variants of S. cerevisiae.


Assuntos
Proteínas Mitocondriais/análise , Proteoma , Proteômica , Proteínas de Saccharomyces cerevisiae/análise , Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas em Tandem , Cromatografia Líquida de Alta Pressão , Regulação Fúngica da Expressão Gênica , Marcação por Isótopo , Proteínas Mitocondriais/genética , Mutação , Projetos de Pesquisa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
19.
Int J Food Microbiol ; 350: 109229, 2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34023682

RESUMO

Amidst rising demand for non-dairy probiotic foods, and growing interest in coffees with added functionalities, it would be opportune to ferment coffee brews with probiotics. However, challenges exist in maintaining probiotic viability in high-moisture food products. Here, we aimed to enhance the viability of the probiotic bacteria, Lactobacillus rhamnosus GG, in coffee brews by co-culturing with the probiotic yeast, Saccharomyces cerevisiae var. boulardii CNCM-I745. The yeast significantly enhanced the viability of L. rhamnosus GG, as bacterial populations beyond 7 Log CFU/mL were maintained throughout 14 weeks of storage at 4 and 25 °C. In contrast, the single culture of L. rhamnosus GG suffered viability losses below 6 Log CFU/mL within 10 weeks at 4 °C, and 3 weeks at 25 °C. Growth and survival of S. boulardii CNCM-I745 remained unaffected by the presence of L. rhamnosus GG. Volatile profiles of coffee brews were altered by probiotic metabolic activities, but co-culturing led to suppressed generation of diacetyl and ethanol compared to single cultures. Probiotic fermentation did not alter principal coffee bioactive compounds and antioxidant capacities; however, declines in peroxyl radical scavenging capacities were observed after ambient storage. Overall, we illustrate that yeasts are effective in enhancing probiotic bacterial viability in coffee brews, which may be useful in developing shelf stable probiotic food products.


Assuntos
Café/microbiologia , Lactobacillus rhamnosus/crescimento & desenvolvimento , Probióticos/metabolismo , Saccharomyces boulardii/crescimento & desenvolvimento , Saccharomyces cerevisiae/crescimento & desenvolvimento , Reatores Biológicos , Café/metabolismo , Fermentação , Lactobacillus rhamnosus/metabolismo , Viabilidade Microbiana , Saccharomyces boulardii/metabolismo , Saccharomyces cerevisiae/metabolismo , Fermento Seco/metabolismo
20.
Biochim Biophys Acta Gen Subj ; 1865(8): 129932, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34022298

RESUMO

Superfluous and damaged mitochondria need to be efficiently repaired or removed. Mitophagy is a selective type of autophagy that can engulf a portion of mitochondria within a double-membrane structure, called a mitophagosome, and deliver it to the vacuole for degradation. Mitophagy has significant physiological functions from yeast to human, and recent advances in yeast mitophagy shed light on the molecular mechanisms of mitophagy, especially the regulation of mitophagy induction. This review summarizes our current knowledge about yeast mitophagy and considers several unsolved questions, with a particular focus on Saccharomyces cerevisiae.


Assuntos
Mitocôndrias/patologia , Mitofagia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Humanos , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
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