ABSTRACT
Cells sense elevated temperatures and mount an adaptive heat shock response that involves changes in gene expression, but the underlying mechanisms, particularly on the level of translation, remain unknown. Here we report that, in budding yeast, the essential translation initiation factor Ded1p undergoes heat-induced phase separation into gel-like condensates. Using ribosome profiling and an in vitro translation assay, we reveal that condensate formation inactivates Ded1p and represses translation of housekeeping mRNAs while promoting translation of stress mRNAs. Testing a variant of Ded1p with altered phase behavior as well as Ded1p homologs from diverse species, we demonstrate that Ded1p condensation is adaptive and fine-tuned to the maximum growth temperature of the respective organism. We conclude that Ded1p condensation is an integral part of an extended heat shock response that selectively represses translation of housekeeping mRNAs to promote survival under conditions of severe heat stress.
Subject(s)
DEAD-box RNA Helicases/metabolism , Gene Expression Regulation, Fungal/genetics , Protein Biosynthesis/genetics , Saccharomyces cerevisiae Proteins/metabolism , DEAD-box RNA Helicases/physiology , Gene Expression/genetics , Genes, Essential/genetics , Heat-Shock Proteins/metabolism , Heat-Shock Response/genetics , RNA, Messenger/metabolism , Ribosomes/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/physiologyABSTRACT
Biological signaling networks use feedback control to dynamically adjust their operation in real time. Traditional static genetic methods such as gene knockouts or rescue experiments can often identify the existence of feedback interactions but are unable to determine what feedback dynamics are required. Here, we implement a new strategy, closed-loop optogenetic compensation (CLOC), to address this problem. Using a custom-built hardware and software infrastructure, CLOC monitors, in real time, the output of a pathway deleted for a feedback regulator. A minimal model uses these measurements to calculate and deliver-on the fly-an optogenetically enabled transcriptional input designed to compensate for the effects of the feedback deletion. Application of CLOC to the yeast pheromone response pathway revealed surprisingly distinct dynamic requirements for three well-studied feedback regulators. CLOC, a marriage of control theory and traditional genetics, presents a broadly applicable methodology for defining the dynamic function of biological feedback regulators.
Subject(s)
Feedback, Physiological , Gene Expression Regulation, Fungal , Optogenetics/methods , Genetic Complementation Test/methods , Mating Factor/genetics , Mating Factor/metabolism , Saccharomyces cerevisiae/genetics , Software , Transcriptional ActivationABSTRACT
To better understand the gene regulatory mechanisms that program developmental processes, we carried out simultaneous genome-wide measurements of mRNA, translation, and protein through meiotic differentiation in budding yeast. Surprisingly, we observed that the levels of several hundred mRNAs are anti-correlated with their corresponding protein products. We show that rather than arising from canonical forms of gene regulatory control, the regulation of at least 380 such cases, or over 8% of all measured genes, involves temporally regulated switching between production of a canonical, translatable transcript and a 5' extended isoform that is not efficiently translated into protein. By this pervasive mechanism for the modulation of protein levels through a natural developmental program, a single transcription factor can coordinately activate and repress protein synthesis for distinct sets of genes. The distinction is not based on whether or not an mRNA is induced but rather on the type of transcript produced.
Subject(s)
Meiosis/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Gene Expression Regulation, Fungal , Genes, Fungal , Models, Biological , Molecular Sequence Annotation , Protein Biosynthesis , Protein Isoforms/genetics , Protein Isoforms/metabolism , Proteome/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproducibility of Results , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/metabolismABSTRACT
The construction of synthetic gene circuits requires the rational combination of multiple regulatory components, but predicting their behavior can be challenging due to poorly understood component interactions and unexpected emergent behaviors. In eukaryotes, chromatin regulators (CRs) are essential regulatory components that orchestrate gene expression. Here, we develop a screening platform to investigate the impact of CR pairs on transcriptional activity in yeast. We construct a combinatorial library consisting of over 1,900 CR pairs and use a high-throughput workflow to characterize the impact of CR co-recruitment on gene expression. We recapitulate known interactions and discover several instances of CR pairs with emergent behaviors. We also demonstrate that supervised machine learning models trained with low-dimensional amino acid embeddings accurately predict the impact of CR co-recruitment on transcriptional activity. This work introduces a scalable platform and machine learning approach that can be used to study how networks of regulatory components impact gene expression.
Subject(s)
Chromatin , Gene Expression Regulation, Fungal , Gene Regulatory Networks , Saccharomyces cerevisiae , Synthetic Biology , Transcription, Genetic , Chromatin/metabolism , Chromatin/genetics , Synthetic Biology/methods , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , High-Throughput Screening Assays/methods , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Supervised Machine Learning , Chromatin Assembly and Disassembly , Transcription Factors/metabolism , Transcription Factors/geneticsABSTRACT
Heat-shocked cells prioritize the translation of heat shock (HS) mRNAs, but the underlying mechanism is unclear. We report that HS in budding yeast induces the disassembly of the eIF4F complex, where eIF4G and eIF4E assemble into translationally arrested mRNA ribonucleoprotein particles (mRNPs) and HS granules (HSGs), whereas eIF4A promotes HS translation. Using in vitro reconstitution biochemistry, we show that a conformational rearrangement of the thermo-sensing eIF4A-binding domain of eIF4G dissociates eIF4A and promotes the assembly with mRNA into HS-mRNPs, which recruit additional translation factors, including Pab1p and eIF4E, to form multi-component condensates. Using extracts and cellular experiments, we demonstrate that HS-mRNPs and condensates repress the translation of associated mRNA and deplete translation factors that are required for housekeeping translation, whereas HS mRNAs can be efficiently translated by eIF4A. We conclude that the eIF4F complex is a thermo-sensing node that regulates translation during HS.
Subject(s)
Eukaryotic Initiation Factor-4F , Eukaryotic Initiation Factor-4G , Heat-Shock Response , Poly(A)-Binding Proteins , Protein Biosynthesis , RNA, Messenger , Ribonucleoproteins , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Heat-Shock Response/genetics , Eukaryotic Initiation Factor-4F/metabolism , Eukaryotic Initiation Factor-4F/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Eukaryotic Initiation Factor-4G/metabolism , Eukaryotic Initiation Factor-4G/genetics , Ribonucleoproteins/metabolism , Ribonucleoproteins/genetics , Eukaryotic Initiation Factor-4E/metabolism , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-4A/metabolism , Eukaryotic Initiation Factor-4A/genetics , Gene Expression Regulation, Fungal , Protein Binding , RNA, Fungal/metabolism , RNA, Fungal/geneticsABSTRACT
In response to stress, eukaryotes activate the integrated stress response (ISR) via phosphorylation of eIF2α to promote the translation of pro-survival effector genes, such as GCN4 in yeast. Complementing the ISR is the target of rapamycin (TOR) pathway, which regulates eIF4E function. Here, we probe translational control in the absence of eIF4E in Saccharomyces cerevisiae. Intriguingly, we find that loss of eIF4E leads to de-repression of GCN4 translation. In addition, we find that de-repression of GCN4 translation is accompanied by neither eIF2α phosphorylation nor reduction in initiator ternary complex (TC). Our data suggest that when eIF4E levels are depleted, GCN4 translation is de-repressed via a unique mechanism that may involve faster scanning by the small ribosome subunit due to increased local concentration of eIF4A. Overall, our findings suggest that relative levels of eIF4F components are key to ribosome dynamics and may play important roles in translational control of gene expression.
Subject(s)
Basic-Leucine Zipper Transcription Factors , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Stress, Physiological , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Phosphorylation , Basic-Leucine Zipper Transcription Factors/metabolism , Basic-Leucine Zipper Transcription Factors/genetics , Eukaryotic Initiation Factor-4F/metabolism , Eukaryotic Initiation Factor-4F/genetics , Protein Biosynthesis , Gene Expression Regulation, Fungal , Eukaryotic Initiation Factor-4E/metabolism , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-2/metabolism , Eukaryotic Initiation Factor-2/genetics , Signal Transduction , Ribosomes/metabolism , Ribosomes/genetics , Eukaryotic Initiation Factor-4A/metabolism , Eukaryotic Initiation Factor-4A/geneticsABSTRACT
Protein synthesis is metabolically costly and must be tightly coordinated with changing cellular needs and nutrient availability. The cap-binding protein eIF4E makes the earliest contact between mRNAs and the translation machinery, offering a key regulatory nexus. We acutely depleted this essential protein and found surprisingly modest effects on cell growth and recovery of protein synthesis. Paradoxically, impaired protein biosynthesis upregulated genes involved in the catabolism of aromatic amino acids simultaneously with the induction of the amino acid biosynthetic regulon driven by the integrated stress response factor GCN4. We further identified the translational control of Pho85 cyclin 5 (PCL5), a negative regulator of Gcn4, that provides a consistent protein-to-mRNA ratio under varied translation environments. This regulation depended in part on a uniquely long poly(A) tract in the PCL5 5' UTR and poly(A) binding protein. Collectively, these results highlight how eIF4E connects protein synthesis to metabolic gene regulation, uncovering mechanisms controlling translation during environmental challenges.
Subject(s)
Amino Acids , Eukaryotic Initiation Factor-4E , Gene Expression Regulation, Fungal , Protein Biosynthesis , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Eukaryotic Initiation Factor-4E/metabolism , Eukaryotic Initiation Factor-4E/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Amino Acids/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , RNA, Messenger/metabolism , RNA, Messenger/genetics , 5' Untranslated Regions , Basic-Leucine Zipper Transcription Factors/metabolism , Basic-Leucine Zipper Transcription Factors/genetics , Cyclins/genetics , Cyclins/metabolism , Poly(A)-Binding Proteins/metabolism , Poly(A)-Binding Proteins/geneticsABSTRACT
Data of gene expression levels across individuals, cell types, and disease states is expanding, yet our understanding of how expression levels impact phenotype is limited. Here, we present a massively parallel system for assaying the effect of gene expression levels on fitness in Saccharomyces cerevisiae by systematically altering the expression level of â¼100 genes at â¼100 distinct levels spanning a 500-fold range at high resolution. We show that the relationship between expression levels and growth is gene and environment specific and provides information on the function, stoichiometry, and interactions of genes. Wild-type expression levels in some conditions are not optimal for growth, and genes whose fitness is greatly affected by small changes in expression level tend to exhibit lower cell-to-cell variability in expression. Our study addresses a fundamental gap in understanding the functional significance of gene expression regulation and offers a framework for evaluating the phenotypic effects of expression variation.
Subject(s)
Gene Expression Regulation, Fungal , Gene-Environment Interaction , Genetic Fitness , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/genetics , DNA Barcoding, Taxonomic , Gene Library , Genes, Fungal , High-Throughput Nucleotide SequencingABSTRACT
Genome-metabolism interactions enable cell growth. To probe the extent of these interactions and delineate their functional contributions, we quantified the Saccharomyces amino acid metabolome and its response to systematic gene deletion. Over one-third of coding genes, in particular those important for chromatin dynamics, translation, and transport, contribute to biosynthetic metabolism. Specific amino acid signatures characterize genes of similar function. This enabled us to exploit functional metabolomics to connect metabolic regulators to their effectors, as exemplified by TORC1, whose inhibition in exponentially growing cells is shown to match an interruption in endomembrane transport. Providing orthogonal information compared to physical and genetic interaction networks, metabolomic signatures cluster more than half of the so far uncharacterized yeast genes and provide functional annotation for them. A major part of coding genes is therefore participating in gene-metabolism interactions that expose the metabolism regulatory network and enable access to an underexplored space in gene function.
Subject(s)
Amino Acids/biosynthesis , Metabolome , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transcription Factors/metabolism , Amino Acids/genetics , Chromatin/metabolism , Gene Deletion , Gene Expression Regulation, Fungal , Gene Regulatory Networks , Metabolome/genetics , Metabolomics/methods , Multigene Family , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/genetics , Transcription, GeneticABSTRACT
Transcription of tRNA genes by RNA polymerase III (RNAPIII) is tuned by signaling cascades. The emerging notion of differential tRNA gene regulation implies the existence of additional regulatory mechanisms. However, tRNA gene-specific regulators have not been described. Decoding the local chromatin proteome of a native tRNA gene in yeast revealed reprogramming of the RNAPIII transcription machinery upon nutrient perturbation. Among the dynamic proteins, we identified Fpt1, a protein of unknown function that uniquely occupied RNAPIII-regulated genes. Fpt1 binding at tRNA genes correlated with the efficiency of RNAPIII eviction upon nutrient perturbation and required the transcription factors TFIIIB and TFIIIC but not RNAPIII. In the absence of Fpt1, eviction of RNAPIII was reduced, and the shutdown of ribosome biogenesis genes was impaired upon nutrient perturbation. Our findings provide support for a chromatin-associated mechanism required for RNAPIII eviction from tRNA genes and tuning the physiological response to changing metabolic demands.
Subject(s)
RNA Polymerase III , Saccharomyces cerevisiae Proteins , RNA Polymerase III/genetics , RNA Polymerase III/metabolism , Proteome/genetics , Proteome/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Chromatin/genetics , Chromatin/metabolism , Gene Expression Regulation, Fungal , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolism , Transcription, GeneticABSTRACT
When mRNAs have been transcribed and processed in the nucleus, they are exported to the cytoplasm for translation. This export is mediated by the export receptor heterodimer Mex67-Mtr2 in the yeast Saccharomyces cerevisiae (TAP-p15 in humans)1,2. Interestingly, many long non-coding RNAs (lncRNAs) also leave the nucleus but it is currently unclear why they move to the cytoplasm3. Here we show that antisense RNAs (asRNAs) accelerate mRNA export by annealing with their sense counterparts through the helicase Dbp2. These double-stranded RNAs (dsRNAs) dominate export compared with single-stranded RNAs (ssRNAs) because they have a higher capacity and affinity for the export receptor Mex67. In this way, asRNAs boost gene expression, which is beneficial for cells. This is particularly important when the expression program changes. Consequently, the degradation of dsRNA, or the prevention of its formation, is toxic for cells. This mechanism illuminates the general cellular occurrence of asRNAs and explains their nuclear export.
Subject(s)
Active Transport, Cell Nucleus , Cell Nucleus , Gene Expression Regulation, Fungal , RNA Transport , RNA, Antisense , RNA, Double-Stranded , RNA, Messenger , Saccharomyces cerevisiae , Cell Nucleus/metabolism , Cytoplasm/metabolism , DEAD-box RNA Helicases/metabolism , DEAD-box RNA Helicases/genetics , Nucleocytoplasmic Transport Proteins/metabolism , Nucleocytoplasmic Transport Proteins/genetics , RNA, Antisense/metabolism , RNA, Antisense/genetics , RNA, Double-Stranded/metabolism , RNA, Double-Stranded/genetics , RNA, Messenger/metabolism , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Acetyl-CoA is a key intermediate situated at the intersection of many metabolic pathways. The reliance of histone acetylation on acetyl-CoA enables the coordination of gene expression with metabolic state. Abundant acetyl-CoA has been linked to the activation of genes involved in cell growth or tumorigenesis through histone acetylation. However, the role of histone acetylation in transcription under low levels of acetyl-CoA remains poorly understood. Here, we use a yeast starvation model to observe the dramatic alteration in the global occupancy of histone acetylation following carbon starvation; the location of histone acetylation marks shifts from growth-promoting genes to gluconeogenic and fat metabolism genes. This reallocation is mediated by both the histone deacetylase Rpd3p and the acetyltransferase Gcn5p, a component of the SAGA transcriptional coactivator. Our findings reveal an unexpected switch in the specificity of histone acetylation to promote pathways that generate acetyl-CoA for oxidation when acetyl-CoA is limiting.
Subject(s)
Gluconeogenesis , Glucose/deficiency , Histones/metabolism , Lipid Metabolism , Protein Processing, Post-Translational , Saccharomyces cerevisiae/metabolism , Acetyl Coenzyme A/metabolism , Acetylation , Gene Expression Regulation, Fungal , Histone Acetyltransferases/genetics , Histone Acetyltransferases/metabolism , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Lipid Metabolism/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Trans-Activators/genetics , Trans-Activators/metabolismABSTRACT
Previous structural studies of the initiation-elongation transition of RNA polymerase II (pol II) transcription have relied on the use of synthetic oligonucleotides, often artificially discontinuous to capture pol II in the initiating state. Here, we report multiple structures of initiation complexes converted de novo from a 33-subunit yeast pre-initiation complex (PIC) through catalytic activities and subsequently stalled at different template positions. We determine that PICs in the initially transcribing complex (ITC) can synthesize a transcript of â¼26 nucleotides before transitioning to an elongation complex (EC) as determined by the loss of general transcription factors (GTFs). Unexpectedly, transition to an EC was greatly accelerated when an ITC encountered a downstream EC stalled at promoter proximal regions and resulted in a collided head-to-end dimeric EC complex. Our structural analysis reveals a dynamic state of TFIIH, the largest of GTFs, in PIC/ITC with distinct functional consequences at multiple steps on the pathway to elongation.
Subject(s)
RNA Polymerase II/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Transcription Initiation, Genetic , Cryoelectron Microscopy , Gene Expression Regulation, Fungal , Models, Molecular , Promoter Regions, Genetic , Protein Conformation , RNA Polymerase II/genetics , RNA Polymerase II/ultrastructure , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/ultrastructure , Structure-Activity Relationship , Time Factors , Transcription Elongation, Genetic , Transcription Factors, TFII/genetics , Transcription Factors, TFII/metabolismABSTRACT
To understand regulatory systems, it would be useful to uniformly determine how different components contribute to the expression of all other genes. We therefore monitored mRNA expression genome-wide, for individual deletions of one-quarter of yeast genes, focusing on (putative) regulators. The resulting genetic perturbation signatures reflect many different properties. These include the architecture of protein complexes and pathways, identification of expression changes compatible with viability, and the varying responsiveness to genetic perturbation. The data are assembled into a genetic perturbation network that shows different connectivities for different classes of regulators. Four feed-forward loop (FFL) types are overrepresented, including incoherent type 2 FFLs that likely represent feedback. Systematic transcription factor classification shows a surprisingly high abundance of gene-specific repressors, suggesting that yeast chromatin is not as generally restrictive to transcription as is often assumed. The data set is useful for studying individual genes and for discovering properties of an entire regulatory system.
Subject(s)
Gene Expression Regulation, Fungal , Gene Regulatory Networks , Genetic Techniques , Saccharomyces cerevisiae/genetics , Transcriptome , Gene Deletion , Gene Knockout TechniquesABSTRACT
The transcription of genomic information in eukaryotes is regulated in large part by chromatin. How a diverse array of chromatin regulator (CR) proteins with different functions and genomic localization patterns coordinates chromatin activity to control transcription remains unclear. Here, we take a synthetic biology approach to decipher the complexity of chromatin regulation by studying emergent transcriptional behaviors from engineered combinatorial, spatial, and temporal patterns of individual CRs. We fuse 223 yeast CRs to programmable zinc finger proteins. Site-specific and combinatorial recruitment of CRs to distinct intralocus locations reveals a range of transcriptional logic and behaviors, including synergistic activation, long-range and spatial regulation, and gene expression memory. Comparing these transcriptional behaviors with annotated CR complex and function terms provides design principles for the engineering of transcriptional regulation. This work presents a bottom-up approach to investigating chromatin-mediated transcriptional regulation and introduces chromatin-based components and systems for synthetic biology and cellular engineering.
Subject(s)
Gene Expression Regulation, Fungal , Gene Regulatory Networks , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Chromatin/metabolism , Genes, Reporter , Saccharomyces cerevisiae Proteins/chemistry , Transcription, GeneticABSTRACT
In addition to their annotated transcript, many eukaryotic mRNA promoters produce divergent noncoding transcripts. To define determinants of divergent promoter directionality, we used genomic replacement experiments. Sequences within noncoding transcripts specified their degradation pathways, and functional protein-coding transcripts could be produced in the divergent direction. To screen for mutants affecting the ratio of transcription in each direction, a bidirectional fluorescent protein reporter construct was introduced into the yeast nonessential gene deletion collection. We identified chromatin assembly as an important regulator of divergent transcription. Mutations in the CAF-I complex caused genome-wide derepression of nascent divergent noncoding transcription. In opposition to the CAF-I chromatin assembly pathway, H3K56 hyperacetylation, together with the nucleosome remodeler SWI/SNF, facilitated divergent transcription by promoting rapid nucleosome turnover. We propose that these chromatin-mediated effects control divergent transcription initiation, complementing downstream pathways linked to early termination and degradation of the noncoding RNAs.
Subject(s)
Chromatin Assembly Factor-1/metabolism , Chromatin/metabolism , Gene Expression Regulation, Fungal , RNA, Fungal/genetics , RNA, Untranslated/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Chromatin Assembly and Disassembly , Nucleosomes/metabolism , Promoter Regions, Genetic , RNA Stability , RNA, Fungal/metabolism , RNA, Untranslated/metabolism , Transcription Termination, Genetic , Transcription, GeneticABSTRACT
Temperature is a variable component of the environment, and all organisms must deal with or adapt to temperature change. Acute temperature change activates cellular stress responses, resulting in refolding or removal of damaged proteins. However, how organisms adapt to long-term temperature change remains largely unexplored. Here we report that budding yeast responds to long-term high temperature challenge by switching from chaperone induction to reduction of temperature-sensitive proteins and re-localizing a portion of its proteome. Surprisingly, we also find that many proteins adopt an alternative conformation. Using Fet3p as an example, we find that the temperature-dependent conformational difference is accompanied by distinct thermostability, subcellular localization, and, importantly, cellular functions. We postulate that, in addition to the known mechanisms of adaptation, conformational plasticity allows some polypeptides to acquire new biophysical properties and functions when environmental change endures.
Subject(s)
Adaptation, Physiological/genetics , Proteome/genetics , Stress, Physiological/genetics , Transcriptome/genetics , Acclimatization/genetics , Animals , Environmental Exposure/adverse effects , Gene Expression Regulation, Fungal/genetics , Hot Temperature/adverse effects , Saccharomycetales/geneticsABSTRACT
mRNA translation is coupled to multiprotein complex assembly in the cytoplasm or to protein delivery into intracellular compartments. Here, by combining systematic RNA immunoprecipitation and single-molecule RNA imaging in yeast, we have provided a complete depiction of the co-translational events involved in the biogenesis of a large multiprotein assembly, the nuclear pore complex (NPC). We report that binary interactions between NPC subunits can be established during translation, in the cytoplasm. Strikingly, the nucleoporins Nup1/Nup2, together with a number of nuclear proteins, are instead translated at nuclear pores, through a mechanism involving interactions between their nascent N-termini and nuclear transport receptors. Uncoupling this co-translational recruitment further triggers the formation of cytoplasmic foci of unassembled polypeptides. Altogether, our data reveal that distinct, spatially segregated modes of co-translational interactions foster the ordered assembly of NPC subunits and that localized translation can ensure the proper delivery of proteins to the pore and the nucleus.
Subject(s)
Nuclear Pore Complex Proteins/genetics , Protein Biosynthesis , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Active Transport, Cell Nucleus , Cytoplasm/genetics , Cytoplasm/metabolism , Gene Expression Regulation, Fungal , Karyopherins/genetics , Karyopherins/metabolism , Nuclear Pore/genetics , Nuclear Pore/metabolism , Nuclear Pore Complex Proteins/classification , Nuclear Pore Complex Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/classification , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Autophagy is a conserved intracellular degradation pathway exerting various cytoprotective and homeostatic functions by using de novo double-membrane vesicle (autophagosome) formation to target a wide range of cytoplasmic material for vacuolar/lysosomal degradation. The Atg1 kinase is one of its key regulators, coordinating a complex signaling program to orchestrate autophagosome formation. Combining in vitro reconstitution and cell-based approaches, we demonstrate that Atg1 is activated by lipidated Atg8 (Atg8-PE), stimulating substrate phosphorylation along the growing autophagosomal membrane. Atg1-dependent phosphorylation of Atg13 triggers Atg1 complex dissociation, enabling rapid turnover of Atg1 complex subunits at the pre-autophagosomal structure (PAS). Moreover, Atg1 recruitment by Atg8-PE self-regulates Atg8-PE levels in the growing autophagosomal membrane by phosphorylating and thus inhibiting the Atg8-specific E2 and E3. Our work uncovers the molecular basis for positive and negative feedback imposed by Atg1 and how opposing phosphorylation and dephosphorylation events underlie the spatiotemporal regulation of autophagy.
Subject(s)
Autophagosomes/enzymology , Autophagy-Related Proteins/metabolism , Autophagy , Protein Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Autophagosomes/genetics , Autophagy-Related Protein 8 Family/genetics , Autophagy-Related Protein 8 Family/metabolism , Autophagy-Related Proteins/genetics , Enzyme Activation , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Fungal , Phosphorylation , Protein Kinases/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Signal Transduction , Time FactorsABSTRACT
Biosynthesis scales with cell size such that protein concentrations generally remain constant as cells grow. As an exception, synthesis of the cell-cycle inhibitor Whi5 "sub-scales" with cell size so that its concentration is lower in larger cells to promote cell-cycle entry. Here, we find that transcriptional control uncouples Whi5 synthesis from cell size, and we identify histones as the major class of sub-scaling transcripts besides WHI5 by screening for similar genes. Histone synthesis is thereby matched to genome content rather than cell size. Such sub-scaling proteins are challenged by asymmetric cell division because proteins are typically partitioned in proportion to newborn cell volume. To avoid this fate, Whi5 uses chromatin-binding to partition similar protein amounts to each newborn cell regardless of cell size. Disrupting both Whi5 synthesis and chromatin-based partitioning weakens G1 size control. Thus, specific transcriptional and partitioning mechanisms determine protein sub-scaling to control cell size.