RESUMO
Although protein aggregation can cause cytotoxicity, such aggregates can also form to mitigate cytotoxicity from misfolded proteins, although the nature of these contrasting aggregates remains unclear. We previously found that overproduction (op) of a three green fluorescent protein-linked protein (3×GFP) induces giant aggregates and is detrimental to growth. Here, we investigated the mechanism of growth inhibition by 3×GFP-op using non-aggregative 3×MOX-op as a control in Saccharomyces cerevisiae. The 3×GFP aggregates were induced by misfolding, and 3×GFP-op had higher cytotoxicity than 3×MOX-op because it perturbed the ubiquitin-proteasome system. Static aggregates formed by 3×GFP-op dynamically trapped Hsp70 family proteins (Ssa1 and Ssa2 in yeast), causing the heat-shock response. Systematic analysis of mutants deficient in the protein quality control suggested that 3×GFP-op did not cause a critical Hsp70 depletion and aggregation functioned in the direction of mitigating toxicity. Artificial trapping of essential cell cycle regulators into 3×GFP aggregates caused abnormalities in the cell cycle. In conclusion, the formation of the giant 3×GFP aggregates itself is not cytotoxic, as it does not entrap and deplete essential proteins. Rather, it is productive, inducing the heat-shock response while preventing an overload to the degradation system.
Assuntos
Proteínas de Fluorescência Verde , Proteínas de Choque Térmico HSP70 , Agregados Proteicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteólise , Complexo de Endopeptidases do Proteassoma/metabolismo , Resposta ao Choque Térmico/genética , Dobramento de Proteína , Ciclo Celular/genética , Adenosina TrifosfatasesRESUMO
Overexpression can help life adapt to stressful environments, making an examination of overexpressed genes valuable for understanding stress tolerance mechanisms. However, a systematic study of genes whose overexpression is functionally adaptive (GOFAs) under stress has yet to be conducted. We developed a new overexpression profiling method and systematically identified GOFAs in Saccharomyces cerevisiae under stress (heat, salt, and oxidative). Our results show that adaptive overexpression compensates for deficiencies and increases fitness under stress, like calcium under salt stress. We also investigated the impact of different genetic backgrounds on GOFAs, which varied among three S. cerevisiae strains reflecting differing calcium and potassium requirements for salt stress tolerance. Our study of a knockout collection also suggested that calcium prevents mitochondrial outbursts under salt stress. Mitochondria-enhancing GOFAs were only adaptive when adequate calcium was available and non-adaptive when calcium was deficient, supporting this idea. Our findings indicate that adaptive overexpression meets the cell's needs for maximizing the organism's adaptive capacity in the given environment and genetic context.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Cálcio , Proteínas de Saccharomyces cerevisiae/genética , Mitocôndrias/genética , Patrimônio GenéticoRESUMO
Upon white light illumination, the growth of the budding yeast Saccharomyces cerevisiae was extremely impaired only in the presence of iodide ions, but not fluoride, chloride and bromide ions. Action spectroscopy revealed that the maximum wavelength of the light is around at 373 nm, corresponding to the UVA region. Using a genetic approach, several genes, including OPY1, HEM1, and PAU11, were identified as suppressors of this growth inhibition. This iodide-dependent UVA-triggered growth inhibition method, along with its suppressive molecules, would be beneficial for understanding cell growth processes in eukaryotes and can be utilized for medium sterilization using UVA light.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Iodetos , Raios Ultravioleta , Luz , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Proper control of gene expression levels upon various perturbations is a fundamental aspect of cellular robustness. Protein-level dosage compensation is one mechanism buffering perturbations to stoichiometry of multiprotein complexes through accelerated proteolysis of unassembled subunits. Although N-terminal acetylation- and ubiquitin-mediated proteasomal degradation by the Ac/N-end rule pathway enables selective compensation of excess subunits, it is unclear how widespread this pathway contributes to stoichiometry control. Here we report that dosage compensation depends only partially on the Ac/N-end rule pathway. Our analysis of genetic interactions between 18 subunits and 12 quality control factors in budding yeast demonstrated that multiple E3 ubiquitin ligases and N-acetyltransferases are involved in dosage compensation. We find that N-acetyltransferases-mediated compensation is not simply predictable from N-terminal sequence despite their sequence specificity for N-acetylation. We also find that the compensation of Pop3 and Bet4 is due in large part to a minor N-acetyltransferase NatD. Furthermore, canonical NatD substrates histone H2A/H4 were compensated even in its absence, suggesting N-acetylation-independent stoichiometry control. Our study reveals the complexity and robustness of the stoichiometry control system.
Assuntos
Mecanismo Genético de Compensação de Dose/genética , Complexos Multiproteicos/genética , Proteólise , Ubiquitina-Proteína Ligases/genética , Acetilação , Arilamina N-Acetiltransferase/genética , Histona Acetiltransferases/genética , Histonas/genética , Isoenzimas/genética , Processamento de Proteína Pós-Traducional/genética , Saccharomycetales/genética , Ubiquitina/genéticaRESUMO
Aneuploidy-the gain or loss of one or more whole chromosome-typically has an adverse impact on organismal fitness, manifest in conditions such as Down syndrome. A central question is whether aneuploid phenotypes are the consequence of copy number changes of a few especially harmful genes that may be present on the extra chromosome or are caused by copy number alterations of many genes that confer no observable phenotype when varied individually. We used the proliferation defect exhibited by budding yeast strains carrying single additional chromosomes (disomes) to distinguish between the "few critical genes" hypothesis and the "mass action of genes" hypothesis. Our results indicate that subtle changes in gene dosage across a chromosome can have significant phenotypic consequences. We conclude that phenotypic thresholds can be crossed by mass action of copy number changes that, on their own, are benign.
Assuntos
Aneuploidia , Cromossomos Fúngicos/genética , Variações do Número de Cópias de DNA/genética , Dosagem de Genes/genética , Saccharomyces cerevisiae/genética , Proliferação de Células/genética , Fenótipo , Saccharomyces cerevisiae/citologiaRESUMO
Eukaryotic cells are composed of organelles, and each organelle contains proteins that play a role in its function. Therefore, the localization of a protein, especially to organelles, is a clue to infer the function of that protein. In this study, we attempted to identify novel mitochondrially localized proteins in the budding yeast Saccharomyces cerevisiae using a fluorescent protein (GFPdeg) that is rapidly degraded in the cytoplasm. Of the budding yeast proteins predicted to localize to mitochondria by the prediction tool Deeploc-1.0, those with known mitochondrial localization or functional relevance were eliminated, and 95 proteins of unknown function were selected as candidates for analysis. By forced expression of GFPdeg fusion proteins with these proteins and observation of their localization, we identified 35 uncharacterized proteins potentially localized to mitochondria (UPMs) including 8 previously identified proteins that localize to mitochondria. Most of these had no N-terminal mitochondrial localization signal and were evolutionarily young "emerging genes" that exist only in S. cerevisiae. Some of these genes were found to be upregulated during the postdiauxic shift phase when mitochondria are being developed, suggesting that they are actually involved in some mitochondrial function.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Citoplasma/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismoRESUMO
Understanding buffering mechanisms for various perturbations is essential for understanding robustness in cellular systems. Protein-level dosage compensation, which arises when changes in gene copy number do not translate linearly into protein level, is one mechanism for buffering against genetic perturbations. Here, we present an approach to identify genes with dosage compensation by increasing the copy number of individual genes using the genetic tug-of-war technique. Our screen of chromosome I suggests that dosage-compensated genes constitute approximately 10% of the genome and consist predominantly of subunits of multi-protein complexes. Importantly, because subunit levels are regulated in a stoichiometry-dependent manner, dosage compensation plays a crucial role in maintaining subunit stoichiometries. Indeed, we observed changes in the levels of a complex when its subunit stoichiometries were perturbed. We further analyzed compensation mechanisms using a proteasome-defective mutant as well as ribosome profiling, which provided strong evidence for compensation by ubiquitin-dependent degradation but not reduced translational efficiency. Thus, our study provides a systematic understanding of dosage compensation and highlights that this post-translational regulation is a critical aspect of robustness in cellular systems.
Assuntos
Mecanismo Genético de Compensação de Dose , Regulação Fúngica da Expressão Gênica , Proteólise , Saccharomyces cerevisiae/genética , Cromossomos Fúngicos/genética , Dosagem de Genes , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Gene overexpression beyond a permissible limit causes defects in cellular functions. However, the permissible limits of most genes are unclear. Previously, we developed a genetic method designated genetic tug-of-war (gTOW) to measure the copy number limit of overexpression of a target gene. In the current study, we applied gTOW to the analysis of all protein-coding genes in the budding yeast Saccharomyces cerevisiae. We showed that the yeast cellular system was robust against an increase in the copy number by up to 100 copies in >80% of the genes. After frameshift and segmentation analyses, we isolated 115 dosage-sensitive genes (DSGs) with copy number limits of 10 or less. DSGs contained a significant number of genes involved in cytoskeletal organization and intracellular transport. DSGs tended to be highly expressed and to encode protein complex members. We demonstrated that the protein burden caused the dosage sensitivity of highly expressed genes using a gTOW experiment in which the open reading frame was replaced with GFP. Dosage sensitivities of some DSGs were rescued by the simultaneous increase in the copy numbers of partner genes, indicating that stoichiometric imbalances among complexes cause dosage sensitivity. The results obtained in this study will provide basic knowledge about the physiology of chromosomal abnormalities and the evolution of chromosomal composition.
Assuntos
Dosagem de Genes , Genes Fúngicos , Saccharomyces cerevisiae/genética , Expressão Gênica , Redes Reguladoras de Genes , Genoma Fúngico , Anotação de Sequência Molecular , Fases de Leitura Aberta , Mapas de Interação de Proteínas , Saccharomyces cerevisiae/metabolismoRESUMO
Spirotryprostatins, an indole alkaloid class of nonribosomal peptides isolated from Aspergillus fumigatus, are known for their antimitotic activity in tumor cells. Because spirotryprostatins and many other chemically complex spiro-carbon-bearing natural products exhibit useful biological activities, identifying and understanding the mechanism of spiro-carbon biosynthesis is of great interest. Here we report a detailed study of spiro-ring formation in spirotryprostatins from tryprostatins derived from the fumitremorgin biosynthetic pathway, using reactants and products prepared with engineered yeast and fungal strains. Unexpectedly, FqzB, an FAD-dependent monooxygenase from the unrelated fumiquinazoline biosynthetic pathway, catalyzed spiro-carbon formation in spirotryprostatin A via an epoxidation route. Furthermore, FtmG, a cytochrome P450 from the fumitremorgin biosynthetic pathway, was determined to catalyze the spiro-ring formation in spirotryprostatin B. Our results highlight the versatile role of oxygenating enzymes in the biosynthesis of structurally complex natural products and indicate that cross-talk of different biosynthetic pathways allows product diversification in natural product biosynthesis.
Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Piperazinas/química , Compostos de Espiro/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Western Blotting , DNA Fúngico/genética , Modelos Moleculares , Estrutura Molecular , Piperazinas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Compostos de Espiro/metabolismo , Relação Estrutura-AtividadeRESUMO
Cells sense and control the number and quality of their organelles, but the underlying mechanisms of this regulation are not understood. Our recent research in the yeast Saccharomyces cerevisiae has shown that long acyl chain ceramides in the endoplasmic reticulum (ER) membrane and the lipid moiety of glycosylphosphatidylinositol (GPI) anchor determine the sorting of GPI-anchored proteins in the ER. Here, we show that a mutant strain, which produces shorter ceramides than the wild-type strain, displays a different count of Golgi cisternae. Moreover, deletions of proteins that remodel the lipid portion of GPI anchors resulted in an abnormal number of Golgi cisternae. Thus, our study reveals that protein sorting in the ER plays a critical role in maintaining Golgi biogenesis.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Saccharomycetales/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transporte Proteico , Ceramidas/metabolismo , Glicosilfosfatidilinositóis/metabolismoRESUMO
Cells can maintain their functions despite fluctuations in intracellular parameters, such as protein activities and gene expression levels. This commonly observed biological property of cells is called robustness. On the other hand, these parameters have different limitations, each reflecting the property of the subsystem containing the parameter. The budding yeast cell cycle is quite fragile upon overexpression of CDC14, but is robust upon overexpression of ESP1. The gene products of both CDC14 and ESP1 are regulated by 1ratio1 binding with their inhibitors (Net1 and Pds1), and a mathematical model predicts the extreme fragility of the cell cycle upon overexpression of CDC14 and ESP1 caused by dosage imbalance between these genes. However, it has not been experimentally shown that dosage imbalance causes fragility of the cell cycle. In this study, we measured the quantitative genetic interactions of these genes by performing combinatorial "genetic tug-of-war" experiments. We first showed experimental evidence that dosage imbalance between CDC14 and NET1 causes fragility. We also showed that fragility arising from dosage imbalance between ESP1 and PDS1 is masked by CDH1 and CLB2. The masking function of CLB2 was stabilization of Pds1 by its phosphorylation. We finally modified Chen's model according to our findings. We thus propose that dosage imbalance causes fragility in biological systems.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Fúngicas/genética , Dosagem de Genes , Saccharomycetales/genética , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Genoma Fúngico , Fosforilação , Saccharomycetales/metabolismoRESUMO
Fungal genome sequencing has revealed many genes coding for biosynthetic enzymes, including polyketide synthases and nonribosomal peptide synthetases. However, characterizing these enzymes and identifying the compounds they synthesize remains a challenge, whether the genes are expressed in their original hosts or in more tractable heterologous hosts, such as yeast. Here, we developed a streamlined method for isolating biosynthetic genes from fungal sources and producing bioactive molecules in an engineered Saccharomyces cerevisiae host strain. We used overlap extension PCR and yeast homologous recombination to clone desired fungal polyketide synthase or a nonribosomal peptide synthetase genes (5-20 kb) into a yeast expression vector quickly and efficiently. This approach was used successfully to clone five polyketide synthases and one nonribosomal peptide synthetase, from various fungal species. Subsequent detailed chemical characterizations of the resulting natural products identified six polyketide and two nonribosomal peptide products, one of which was a new compound. Our system should facilitate investigating uncharacterized fungal biosynthetic genes, identifying novel natural products, and rationally engineering biosynthetic pathways for the production of enzyme analogues possessing modified bioactivity.
Assuntos
Genoma Fúngico , Peptídeo Sintases/genética , Policetídeo Sintases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Vias Biossintéticas , Engenharia Genética/métodos , Estrutura Molecular , Peptídeo Sintases/metabolismo , Policetídeo Sintases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Cellular systems are generally robust against fluctuations of intracellular parameters such as gene expression level. However, little is known about expression limits of genes required to halt cellular systems. In this study, using the fission yeast Schizosaccharomyces pombe, we developed a genetic 'tug-of-war' (gTOW) method to assess the overexpression limit of certain genes. Using gTOW, we determined copy number limits for 31 cell-cycle regulators; the limits varied from 1 to >100. Comparison with orthologs of the budding yeast Saccharomyces cerevisiae suggested the presence of a conserved fragile core in the eukaryotic cell cycle. Robustness profiles of networks regulating cytokinesis in both yeasts (septation-initiation network (SIN) and mitotic exit network (MEN)) were quite different, probably reflecting differences in their physiologic functions. Fragility in the regulation of GTPase spg1 was due to dosage imbalance against GTPase-activating protein (GAP) byr4. Using the gTOW data, we modified a mathematical model and successfully reproduced the robustness of the S. pombe cell cycle with the model.
Assuntos
Ciclo Celular/genética , Regulação Fúngica da Expressão Gênica , Modelos Genéticos , Schizosaccharomyces/genética , Proteínas de Ciclo Celular/genética , Simulação por Computador , Citocinese/genética , GTP Fosfo-Hidrolases/genética , Dosagem de Genes , Schizosaccharomyces/citologia , Proteínas de Schizosaccharomyces pombe/genética , Biologia de SistemasRESUMO
The enhanced green fluorescent protein (EGFP) is considered to be a harmless protein because the critical expression level that causes growth defects is higher than that of other proteins. Here, we found that overexpression of EGFP, but not a glycolytic protein Gpm1, triggered the cell elongation phenotype in the budding yeast Saccharomyces cerevisiae. By the morphological analysis of the cell overexpressing fluorescent protein and glycolytic enzyme variants, we revealed that cysteine content was associated with the cell elongation phenotype. The abnormal cell morphology triggered by overexpression of EGFP was also observed in the fission yeast Schizosaccharomyces pombe. Overexpression of cysteine-containing protein was toxic, especially at high-temperature, while the toxicity could be modulated by additional protein characteristics. Investigation of protein aggregate formation, morphological abnormalities in mutants, and transcriptomic changes that occur upon overexpression of EGFP variants suggested that perturbation of the proteasome by the exposed cysteine of the overexpressed protein causes cell elongation. Overexpression of proteins with relatively low folding properties, such as EGFP, was also found to promote the formation of SHOTA (Seventy kDa Heat shock protein-containing, Overexpression-Triggered Aggregates), an intracellular aggregate that incorporates Hsp70/Ssa1, which induces a heat shock response, while it was unrelated to cell elongation. Evolutionary analysis of duplicated genes showed that cysteine toxicity may be an evolutionary bias to exclude cysteine from highly expressed proteins. The overexpression of cysteine-less moxGFP, the least toxic protein revealed in this study, would be a good model system to understand the physiological state of protein burden triggered by ultimate overexpression of harmless proteins.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Cisteína , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
With the accumulation of data on complex molecular machineries coordinating cell-cycle dynamics, coupled with its central function in disease patho-physiologies, it is becoming increasingly important to collate the disparate knowledge sources into a comprehensive molecular network amenable to systems-level analyses. In this work, we present a comprehensive map of the budding yeast cell-cycle, curating reactions from â¼600 original papers. Toward leveraging the map as a framework to explore the underlying network architecture, we abstract the molecular components into three planes--signaling, cell-cycle core and structural planes. The planar view together with topological analyses facilitates network-centric identification of functions and control mechanisms. Further, we perform a comparative motif analysis to identify around 194 motifs including feed-forward, mutual inhibitory and feedback mechanisms contributing to cell-cycle robustness. We envisage the open access, comprehensive cell-cycle map to open roads toward community-based deeper understanding of cell-cycle dynamics.
Assuntos
Ciclo Celular , Saccharomycetales/fisiologia , Motivos de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Modelos Biológicos , Modelos Estatísticos , Fases de Leitura Aberta , Saccharomycetales/genética , Transdução de SinaisRESUMO
Protein overexpression sometimes causes cellular defects, although the underlying mechanism is still unknown. A protein's expression limit, which triggers cellular defects, is a useful indication of the underlying mechanism. In this study, we developed an experimental method of estimating the expression limits of target proteins in the human embryonic kidney cell line HEK293 by measuring the proteins' expression levels in cells that survived after the high-copy introduction of plasmid DNA by which the proteins were expressed under a strong cytomegalovirus promoter. The expression limits of nonfluorescent target proteins were indirectly estimated by measuring the levels of green fluorescent protein (GFP) connected to the target proteins with the self-cleaving sequence P2A. The expression limit of a model GFP was ~5.0% of the total protein, and sustained GFP overexpression caused cell death. The expression limits of GFPs with mitochondria-targeting signals and endoplasmic reticulum localization signals were 1.6% and 0.38%, respectively. The expression limits of four proteins involved in vesicular trafficking were far lower compared to a red fluorescent protein. The protein expression limit estimation method developed will be valuable for defining toxic proteins and consequences of protein overexpression.
Assuntos
Morte Celular/genética , Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Biossíntese de Proteínas/genética , Citomegalovirus/genética , DNA , Retículo Endoplasmático , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Metotrexato , Plasmídeos , Regiões Promotoras Genéticas , Transporte Proteico , Tetra-Hidrofolato Desidrogenase , Proteína Vermelha FluorescenteRESUMO
Overproduction (op) of proteins triggers cellular defects. One of the consequences of overproduction is the protein burden/cost, which is produced by an overloading of the protein synthesis process. However, the physiology of cells under a protein burden is not well characterized. We performed genetic profiling of protein burden by systematic analysis of genetic interactions between GFP-op, surveying both deletion and temperature-sensitive mutants in budding yeast. We also performed genetic profiling in cells with overproduction of triple-GFP (tGFP), and the nuclear export signal-containing tGFP (NES-tGFP). The mutants specifically interacted with GFP-op were suggestive of unexpected connections between actin-related processes like polarization and the protein burden, which was supported by morphological analysis. The tGFP-op interactions suggested that this protein probe overloads the proteasome, whereas those that interacted with NES-tGFP involved genes encoding components of the nuclear export process, providing a resource for further analysis of the protein burden and nuclear export overload.
Assuntos
Transporte Ativo do Núcleo Celular/genética , Sinais de Exportação Nuclear/genética , Complexo de Endopeptidases do Proteassoma , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Núcleo Celular/metabolismo , Perfil Genético , Genômica , Proteínas de Fluorescência Verde , Mutação , Biossíntese de Proteínas/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Silica cell walls of diatoms have attracted attention as a source of nanostructured functional materials and have immense potential for a variety of applications. Previous studies of silica cell wall formation have identified numerous involved proteins, but most of these proteins are species-specific and are not conserved among diatoms. However, because the basic process of diatom cell wall formation is common to all diatom species, ubiquitous proteins and molecules will reveal the mechanisms of cell wall formation. In this study, we assembled de novo transcriptomes of three diatom species, Nitzschia palea, Achnanthes kuwaitensis, and Pseudoleyanella lunata, and compared protein-coding genes of five genome-sequenced diatom species. These analyses revealed a number of diatom-specific genes that encode putative endoplasmic reticulum-targeting proteins. Significant numbers of these proteins showed homology to silicanin-1, which is a conserved diatom protein that reportedly contributes to cell wall formation. These proteins also included a previously unrecognized SET domain protein methyltransferase family that may regulate functions of cell wall formation-related proteins and long-chain polyamines. Proteomic analysis of cell wall-associated proteins in N. palea identified a protein that is also encoded by one of the diatom-specific genes. Expression analysis showed that candidate genes were upregulated in response to silicon, suggesting that these genes play roles in silica cell wall formation. These candidate genes can facilitate further investigations of silica cell wall formation in diatoms.
Assuntos
Parede Celular/metabolismo , Diatomáceas/genética , Diatomáceas/metabolismo , Transcriptoma , Parede Celular/genética , Domínios PR-SET , Proteínas Metiltransferases/metabolismo , Dióxido de Silício/químicaRESUMO
Extreme overproduction of gratuitous proteins can overload cellular protein production resources, leading to growth defects, a phenomenon known as the protein burden/cost effect. Genetic screening in the budding yeast Saccharomyces cerevisiae has isolated several dubious ORFs whose deletions mitigated the protein burden effect, but individual characterization thereof has yet to be delineated. We found that deletion of the YJL175W ORF yielded an N-terminal deletion of Swi3, a subunit of the SWI/SNF chromatin remodeling complex, and partial loss of function of Swi3. The deletion mutant showed a reduction in transcription of genes encoding highly expressed, secreted proteins and an overall reduction in translation. Mutations in the chromatin remodeling complex could thus mitigate the protein burden effect, likely by reallocating residual cellular resources used to overproduce proteins. This cellular state might also be related to cancer cells, as they frequently harbor mutations in the SWI/SNF complex.
Assuntos
Proteínas Nucleares/genética , Fases de Leitura Aberta/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Deleção de Sequência , RNA Mensageiro/genética , Transcrição GênicaRESUMO
Intracellular biochemical parameters, such as the expression level of gene products, are considered to be optimized so that a biological system, including the parameters, works effectively. Those parameters should have some permissible range so that the systems have robustness against perturbations, such as noise in gene expression. However, little is known about the permissible range in real cells because there has been no experimental technique to test it. In this study, we developed a genetic screening method, named "genetic tug-of-war" (gTOW) that evaluates upper limit copy numbers of genes in a model eukaryote Saccharomyces cerevisiae, and we applied it for 30 cell-cycle related genes (CDC genes). The experiment provided unique quantitative data that could be used to argue the system-level properties of the cell cycle such as robustness and fragility. The data were used to evaluate the current computational model, and refinements to the model were suggested.