RESUMO
Stalled protein synthesis produces defective nascent chains that can harm cells. In response, cells degrade these nascent chains via a process called ribosome-associated quality control (RQC). Here, we review the irregularities in the translation process that cause ribosomes to stall as well as how cells use RQC to detect stalled ribosomes, ubiquitylate their tethered nascent chains, and deliver the ubiquitylated nascent chains to the proteasome. We additionally summarize how cells respond to RQC failure.
Assuntos
Escherichia coli/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Ribossomos/genética , Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Poli A/química , Poli A/genética , Poli A/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteólise , Splicing de RNA , Estabilidade de RNA , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Cellular functioning requires the orchestration of thousands of molecular interactions in time and space. Yet most molecules in a cell move by diffusion, which is sensitive to external factors like temperature. How cells sustain complex, diffusion-based systems across wide temperature ranges is unknown. Here, we uncover a mechanism by which budding yeast modulate viscosity in response to temperature and energy availability. This "viscoadaptation" uses regulated synthesis of glycogen and trehalose to vary the viscosity of the cytosol. Viscoadaptation functions as a stress response and a homeostatic mechanism, allowing cells to maintain invariant diffusion across a 20°C temperature range. Perturbations to viscoadaptation affect solubility and phase separation, suggesting that viscoadaptation may have implications for multiple biophysical processes in the cell. Conditions that lower ATP trigger viscoadaptation, linking energy availability to rate regulation of diffusion-controlled processes. Viscoadaptation reveals viscosity to be a tunable property for regulating diffusion-controlled processes in a changing environment.
Assuntos
Metabolismo Energético , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Temperatura , Adaptação Fisiológica , Trifosfato de Adenosina/metabolismo , Difusão , Glicogênio/metabolismo , Homeostase , Modelos Biológicos , Solubilidade , Trealose , ViscosidadeRESUMO
C-terminal tailing is an ancient and conserved form of peptide synthesis that protects cells from incomplete and potentially toxic translation products. Filbeck et al. (2020) and Crowe-McAuliffe et al. (2020) use structural, genetic, and biochemical approaches to elucidate the mechanisms driving C-terminal tailing.
Assuntos
Bactérias , Ribossomos , Controle de QualidadeRESUMO
The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.
Assuntos
Proteínas de Bactérias/genética , Marcação de Genes/métodos , Streptococcus pyogenes , Células HEK293 , Células HeLa , Humanos , Saccharomyces cerevisiae/genética , Pequeno RNA não TraduzidoRESUMO
We present a genetic interaction map of pairwise measures including â¼40% of nonessential S. pombe genes. By comparing interaction maps for fission and budding yeast, we confirmed widespread conservation of genetic relationships within and between complexes and pathways. However, we identified an important subset of orthologous complexes that have undergone functional "repurposing": the evolution of divergent functions and partnerships. We validated three functional repurposing events in S. pombe and mammalian cells and discovered that (1) two lumenal sensors of misfolded ER proteins, the kinase/nuclease Ire1 and the glucosyltransferase Gpt1, act together to mount an ER stress response; (2) ESCRT factors regulate spindle-pole-body duplication; and (3) a membrane-protein phosphatase and kinase complex, the STRIPAK complex, bridges the cis-Golgi, the centrosome, and the outer nuclear membrane to direct mitotic progression. Each discovery opens new areas of inquiry and-together-have implications for model organism-based research and the evolution of genetic systems.
Assuntos
Epistasia Genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Evolução Biológica , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Glicoproteínas de Membrana , Mitose , Complexos Multiproteicos/metabolismo , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Fuso Acromático , Resposta a Proteínas não DobradasRESUMO
The conserved transcriptional regulator heat shock factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol. We surveyed Hsf1 activity in a genome-wide loss-of-function library in Saccaromyces cerevisiae as well as ~78,000 double mutants and found Hsf1 activity to be modulated by highly diverse stresses. These included disruption of a ribosome-bound complex we named the Ribosome Quality Control Complex (RQC) comprising the Ltn1 E3 ubiquitin ligase, two highly conserved but poorly characterized proteins (Tae2 and Rqc1), and Cdc48 and its cofactors. Electron microscopy and biochemical analyses revealed that the RQC forms a stable complex with 60S ribosomal subunits containing stalled polypeptides and triggers their degradation. A negative feedback loop regulates the RQC, and Hsf1 senses an RQC-mediated translation-stress signal distinctly from other stresses. Our work reveals the range of stresses Hsf1 monitors and elucidates a conserved cotranslational protein quality control mechanism.
Assuntos
Complexos Multiproteicos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Choque Térmico/genética , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Ligação a RNA , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteína com ValosinaRESUMO
Mitochondrial dysfunction and proteostasis failure frequently coexist as hallmarks of neurodegenerative disease. How these pathologies are related is not well understood. Here, we describe a phenomenon termed MISTERMINATE (mitochondrial-stress-induced translational termination impairment and protein carboxyl terminal extension), which mechanistically links mitochondrial dysfunction with proteostasis failure. We show that mitochondrial dysfunction impairs translational termination of nuclear-encoded mitochondrial mRNAs, including complex-I 30kD subunit (C-I30) mRNA, occurring on the mitochondrial surface in Drosophila and mammalian cells. Ribosomes stalled at the normal stop codon continue to add to the C terminus of C-I30 certain amino acids non-coded by mRNA template. C-terminally extended C-I30 is toxic when assembled into C-I and forms aggregates in the cytosol. Enhancing co-translational quality control prevents C-I30 C-terminal extension and rescues mitochondrial and neuromuscular degeneration in a Parkinson's disease model. These findings emphasize the importance of efficient translation termination and reveal unexpected link between mitochondrial health and proteome homeostasis mediated by MISTERMINATE.
Assuntos
Códon de Terminação , Proteínas de Drosophila/metabolismo , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Deficiências na Proteostase/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Células HeLa , Humanos , Mitocôndrias/genética , Mitocôndrias/patologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/genética , Deficiências na Proteostase/genética , Deficiências na Proteostase/patologia , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismoRESUMO
Diverse perturbations to endoplasmic reticulum (ER) functions compromise the proper folding and structural maturation of secretory proteins. To study secretory pathway physiology during such "ER stress," we employed an ER-targeted, redox-responsive, green fluorescent protein-eroGFP-that reports on ambient changes in oxidizing potential. Here we find that diverse ER stress regimes cause properly folded, ER-resident eroGFP (and other ER luminal proteins) to "reflux" back to the reducing environment of the cytosol as intact, folded proteins. By utilizing eroGFP in a comprehensive genetic screen in Saccharomyces cerevisiae, we show that ER protein reflux during ER stress requires specific chaperones and cochaperones residing in both the ER and the cytosol. Chaperone-mediated ER protein reflux does not require E3 ligase activity, and proceeds even more vigorously when these ER-associated degradation (ERAD) factors are crippled, suggesting that reflux may work in parallel with ERAD. In summary, chaperone-mediated ER protein reflux may be a conserved protein quality control process that evolved to maintain secretory pathway homeostasis during ER protein-folding stress.
Assuntos
Citosol/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/metabolismo , Chaperonas Moleculares/metabolismo , Degradação Associada com o Retículo Endoplasmático/fisiologia , Homeostase/fisiologia , Oxirredução , Dobramento de Proteína , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Premature arrest of protein synthesis within the open reading frame elicits a protective response that degrades the incomplete nascent chain. In this response, arrested 80S ribosomes are split into their large and small subunits, allowing assembly of the ribosome quality control complex (RQC), which targets nascent chains for degradation. How the cell recognizes arrested nascent chains among the vast pool of actively translating polypeptides is poorly understood. We systematically examined translation arrest and modification of nascent chains in Saccharomyces cerevisiae to characterize the steps that couple arrest to RQC targeting. We focused our analysis on two poorly understood 80S ribosome-binding proteins previously implicated in the response to failed translation, Asc1 and Hel2, as well as a new component of the pathway, Slh1, that we identified here. We found that premature arrest at ribosome stalling sequences still occurred robustly in the absence of Asc1, Hel2, and Slh1. However, these three factors were required for the RQC to modify the nascent chain. We propose that Asc1, Hel2, and Slh1 target arresting ribosomes and that this targeting event is a precondition for the RQC to engage the incomplete nascent chain and facilitate its degradation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , RNA Helicases DEAD-box/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcriptoma , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The ribosome-associated quality control (RQC) pathway resolves stalled ribosomes. As part of RQC, stalled nascent polypeptide chains (NCs) are appended with CArboxy-Terminal amino acids (CAT tails) in an mRNA-free, non-canonical elongation process. CAT tail composition includes Ala, Thr, and potentially other residues. The relationship between CAT tail composition and function has remained unknown. Using biochemical approaches in yeast, we discovered that mechanochemical forces on the NC regulate CAT tailing. We propose CAT tailing initially operates in an "extrusion mode" that increases NC lysine accessibility for on-ribosome ubiquitination. Thr in CAT tails enhances NC extrusion by preventing formation of polyalanine, which can form α-helices. After NC ubiquitylation, pulling forces on the NC switch CAT tailing to an Ala-only "release mode" which facilitates nascent chain release from large ribosomal subunits and NC degradation. Failure to switch from extrusion to release mode leads to accumulation of NCs on large ribosomal subunits and proteotoxic aggregation of Thr-rich CAT tails.
RESUMO
While inhomogeneous diffusivity has been identified as a ubiquitous feature of the cellular interior, its implications for particle mobility and concentration at different length scales remain largely unexplored. In this work, we use agent-based simulations of diffusion to investigate how heterogeneous diffusivity affects the movement and concentration of diffusing particles. We propose that a nonequilibrium mode of membrane-less compartmentalization arising from the convergence of diffusive trajectories into low-diffusive sinks, which we call 'diffusive lensing,' is relevant for living systems. Our work highlights the phenomenon of diffusive lensing as a potentially key driver of mesoscale dynamics in the cytoplasm, with possible far-reaching implications for biochemical processes.
Assuntos
Citoplasma , Difusão , Transporte Biológico , Citoplasma/metabolismo , Modelos Biológicos , Compartimento Celular , Simulação por ComputadorRESUMO
Translational control exerts immediate effect on the composition, abundance, and integrity of the proteome. Ribosome-associated quality control (RQC) handles ribosomes stalled at the elongation and termination steps of translation, with ZNF598 in mammals and Hel2 in yeast serving as key sensors of translation stalling and coordinators of downstream resolution of collided ribosomes, termination of stalled translation, and removal of faulty translation products. The physiological regulation of RQC in general and ZNF598 in particular in multicellular settings is underexplored. Here we show that ZNF598 undergoes regulatory K63-linked ubiquitination in a CNOT4-dependent manner and is upregulated upon mitochondrial stresses in mammalian cells and Drosophila. ZNF598 promotes resolution of stalled ribosomes and protects against mitochondrial stress in a ubiquitination-dependent fashion. In Drosophila models of neurodegenerative diseases and patient cells, ZNF598 overexpression aborts stalled translation of mitochondrial outer membrane-associated mRNAs, removes faulty translation products causal of disease, and improves mitochondrial and tissue health. These results shed lights on the regulation of ZNF598 and its functional role in mitochondrial and tissue homeostasis.
Assuntos
Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae , Animais , Humanos , Proteínas de Transporte/metabolismo , Drosophila/metabolismo , Homeostase , Mamíferos/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
In budding yeast, the transcriptional repressor Opi1 regulates phospholipid biosynthesis by repressing expression of genes containing inositol-sensitive upstream activation sequences. Upon genotoxic stress, cells activate the DNA damage response to coordinate a complex network of signaling pathways aimed at preserving genomic integrity. Here, we reveal that Opi1 is important to modulate transcription in response to genotoxic stress. We find that cells lacking Opi1 exhibit hypersensitivity to genotoxins, along with a delayed G1-to-S-phase transition and decreased gamma-H2A levels. Transcriptome analysis using RNA sequencing reveals that Opi1 plays a central role in modulating essential biological processes during methyl methanesulfonate (MMS)-associated stress, including repression of phospholipid biosynthesis and transduction of mating signaling. Moreover, Opi1 induces sulfate assimilation and amino acid metabolic processes, such as arginine and histidine biosynthesis and glycine catabolism. Furthermore, we observe increased mitochondrial DNA instability in opi1Δ cells upon MMS treatment. Notably, we show that constitutive activation of the transcription factor Ino2-Ino4 is responsible for genotoxin sensitivity in Opi1-deficient cells, and the production of inositol pyrophosphates by Kcs1 counteracts Opi1 function specifically during MMS-induced stress. Overall, our findings highlight Opi1 as a critical sensor of genotoxic stress in budding yeast, orchestrating gene expression to facilitate appropriate stress responses.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Dano ao DNA , Regulação Fúngica da Expressão Gênica , Inositol/metabolismo , Inositol/farmacologia , Fosfolipídeos/metabolismo , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Fatores de Transcrição/genéticaRESUMO
Platinum (Pt) compounds such as oxaliplatin are among the most commonly prescribed anti-cancer drugs. Despite their considerable clinical impact, the molecular basis of platinum cytotoxicity and cancer specificity remain unclear. Here we show that oxaliplatin, a backbone for the treatment of colorectal cancer, causes liquid-liquid demixing of nucleoli at clinically relevant concentrations. Our data suggest that this biophysical defect leads to cell-cycle arrest, shutdown of Pol I-mediated transcription, and ultimately cell death. We propose that instead of targeting a single molecule, oxaliplatin preferentially partitions into nucleoli, where it modifies nucleolar RNA and proteins. This mechanism provides a general approach for drugging the increasing number of cellular processes linked to biomolecular condensates.
Assuntos
Antineoplásicos , Platina , Oxaliplatina/farmacologia , Platina/metabolismo , Nucléolo Celular/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/metabolismo , RNA Polimerase I/metabolismoRESUMO
Aging, disease, and environmental stressors are associated with failures in the ubiquitin-proteasome system (UPS), yet a quantitative understanding of how stressors affect the proteome and how the UPS responds is lacking. Here we assessed UPS performance and adaptability in yeast under stressors using quantitative measurements of misfolded substrate stability and stress-dependent UPS regulation by the transcription factor Rpn4. We found that impairing degradation rates (proteolytic stress) and generating misfolded proteins (folding stress) elicited distinct effects on the proteome and on UPS adaptation. Folding stressors stabilized proteins via aggregation rather than overburdening the proteasome, as occurred under proteolytic stress. Still, the UPS productively adapted to both stressors using separate mechanisms: proteolytic stressors caused Rpn4 stabilization while folding stressors increased RPN4 transcription. In some cases, adaptation completely prevented loss of UPS substrate degradation. Our work reveals the distinct effects of proteotoxic stressors and the versatility of cells in adapting the UPS.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Proteólise , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Resposta ao Choque Térmico , Agregados Proteicos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico , Especificidade por Substrato , Transcrição Gênica , Resposta a Proteínas não DobradasRESUMO
The heat shock response (HSR) is a gene expression program that protects cells from heat and proteotoxic stressors. In this issue, Feder et al. (2020. J. Cell Biol.https://doi.org/10.1083/jcb.202005165) show that subcellular relocalization of the cochaperone Sis1 drives the HSR by de-suppressing the transcription factor Hsf1.
Assuntos
Resposta ao Choque Térmico , Fatores de Transcrição , Regulação da Expressão Gênica , Fatores de Transcrição de Choque Térmico/genética , Fatores de Transcrição de Choque Térmico/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Understanding cellular stress response pathways is challenging because of the complexity of regulatory mechanisms and response dynamics, which can vary with both time and the type of stress. We developed a reverse genetic method called ReporterSeq to comprehensively identify genes regulating a stress-induced transcription factor under multiple conditions in a time-resolved manner. ReporterSeq links RNA-encoded barcode levels to pathway-specific output under genetic perturbations, allowing pooled pathway activity measurements via DNA sequencing alone and without cell enrichment or single-cell isolation. We used ReporterSeq to identify regulators of the heat shock response (HSR), a conserved, poorly understood transcriptional program that protects cells from proteotoxicity and is misregulated in disease. Genome-wide HSR regulation in budding yeast was assessed across 15 stress conditions, uncovering novel stress-specific, time-specific, and constitutive regulators. ReporterSeq can assess the genetic regulators of any transcriptional pathway with the scale of pooled genetic screens and the precision of pathway-specific readouts.
Assuntos
Regulação Fúngica da Expressão Gênica/genética , Genoma Fúngico/fisiologia , Resposta ao Choque Térmico/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/metabolismo , Genética Reversa , Saccharomyces cerevisiae/genéticaRESUMO
Stalled mRNA translation results in the production of incompletely synthesized proteins that are targeted for degradation by ribosome-associated quality control (RQC). Here we investigated the fate of defective proteins translated from stall-inducing, nonstop mRNA that escape ubiquitylation by the RQC protein LTN1. We found that nonstop protein products accumulated in nucleoli and this localization was driven by polylysine tracts produced by translation of the poly(A) tails of nonstop mRNA. Nucleolar sequestration increased the solubility of invading proteins but disrupted nucleoli, altering their dynamics, morphology, and resistance to stress in cell culture and intact flies. Our work elucidates how stalled translation may affect distal cellular processes and may inform studies on the pathology of diseases caused by failures in RQC and characterized by nucleolar stress.
Assuntos
Homeostase/fisiologia , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Humanos , Biossíntese de Proteínas/fisiologia , Ribossomos/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitinação/fisiologiaRESUMO
The Ribosome-associated Quality Control (RQC) pathway co-translationally marks incomplete polypeptides from stalled translation with two signals that trigger their proteasome-mediated degradation. The E3 ligase Ltn1 adds ubiquitin and Rqc2 directs the large ribosomal subunit to append carboxy-terminal alanine and threonine residues (CAT tails). When excessive amounts of incomplete polypeptides evade Ltn1, CAT-tailed proteins accumulate and can self-associate into aggregates. CAT tail aggregation has been hypothesized to either protect cells by sequestering potentially toxic incomplete polypeptides or harm cells by disrupting protein homeostasis. To distinguish between these possibilities, we modulated CAT tail aggregation in Saccharomyces cerevisiae with genetic and chemical tools to analyze CAT tails in aggregated and un-aggregated states. We found that enhancing CAT tail aggregation induces proteotoxic stress and antagonizes degradation of CAT-tailed proteins, while inhibiting aggregation reverses these effects. Our findings suggest that CAT tail aggregation harms RQC-compromised cells and that preventing aggregation can mitigate this toxicity.