Saccharomyces cerevisiae
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA362545 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR5188590.fastq.gz | Fastqsanger.gz | |||
| SRR5188600.fastq.gz | Fastqsanger.gz | |||
| SRR5188610.fastq.gz | Fastqsanger.gz | |||
| SRR5188620.fastq.gz | Fastqsanger.gz | |||
| SRR5188591.fastq.gz | Fastqsanger.gz |
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Project description:The yeast Hsp70 chaperone Ssb interacts with ribosomes and nascent chains to co-translationally assist protein folding. Here, we present a proteome-wide analysis of Hsp70 function during translation, based on in vivo selective ribosome profiling, that reveals mechanistic principles coordinating translation with chaperone-assisted protein folding. Ssb binds most cytosolic, nuclear, and mitochondrial proteins and a subset of ER proteins, supporting its general chaperone function. Position-resolved analysis of Ssb engagement reveals compartment- and protein-specific nascent chain binding profiles that are coordinated by emergence of positively charged peptide stretches enriched in aromatic amino acids. Ssbs’ function is temporally coordinated by RAC but independent from NAC. Analysis of ribosome footprint densities along orfs reveals that ribosomes translate faster at times of Ssb binding. This is coordinated by biases in mRNA secondary structure, and codon usage as well as the action of Ssb, suggesting chaperones may allow higher protein synthesis rates by actively coordinating protein synthesis with co-translational folding.
2017-07-17 | GSE93830 | GEO
Project description:Folding newly synthesized proteins relies on the ribosome intricately coordinating mRNA translation with a network of ribosome-associated machinery. The principles that drive the coordination of this diverse machinery remain poorly understood. Here, we use selective ribosome profiling to determine how the essential chaperonin TRiC/CCT and the Hsp70 Ssb are recruited to ribosome-nascent chain complexes to mediate cotranslational protein folding. Whereas substrate localization and nascent chain sequence are the major determinants of cotranslational recruitment of Ssb, we found that temporal and structural elements drive TRiC engagement. For both chaperones, however, local slowdowns in translation enhance chaperone enrichment. This work helps define the principles that dictate the coordinated activity of ribosome-associated factors to perform their critical role in maintaining a properly folded nascent proteome.
2019-08-22 | GSE114882 | GEO
Project description:Correct and efficient folding of nascent proteins to their native state requires support from the protein homeostasis network. We set to examine which newly translated proteins are less thermostable to infer which polypeptides require more time to fold. More specifically, we sought to determine which newly translated proteins are more susceptible to misfolding and aggregation under heat stress using pulse SILAC. These proteins were abundant, shorter, and highly ordered, with a potentially larger hydrophobic core as suggested by their higher hydrophobicity. Notably these proteins contain more β-sheets that typically require more time for folding and were enriched for Hsp70/Ssb and TRiC/CCT binding motifs, suggesting a higher demand for chaperone-assisted folding. These polypeptides were also more often components of stable protein complexes. All evidence combined suggests that a specific subset of newly translated proteins requires more time following synthesis to reach a thermostable native state in the cell.
2022-08-10 | PXD024336 | Pride
Project description:Accurate and efficient folding of nascent protein sequences into their native state requires support from the protein homeostasis network. Herein we probed which newly translated proteins are thermo-sensitive to infer which polypeptides require more time to fold within the proteome. Specifically, we determined which of these proteins were more susceptible to misfolding and aggregation under heat stress using pulse SILAC coupled mass spectrometry. These proteins are abundant, short, and highly structured. Notably these proteins display a tendency to form β-sheet secondary structures, a configuration which typically requires more time for folding, and were enriched for Hsp70/Ssb and TRiC/CCT binding motifs, suggesting a higher demand for chaperone-assisted folding. These polypeptides were also more often components of stable protein complexes in comparison to other proteins. Combining this evidence suggests that a specific subset of newly translated proteins in the cell requires more time following synthesis to reach a state less prone to aggregation upon stress.
2022-08-10 | PXD028934 | Pride
Project description:Folding of newly synthesized proteins to the native state is a major challenge within the crowded cellular environment, since non-productive interactions can lead to misfolding, aggregation and degradation1. Cells cope with this challenge by coupling synthesis with polypeptide folding and by employing molecular chaperones to safeguard folding already cotranslationally2. However, little is known about the final step of folding, the assembly of polypeptides into complexes, although most of the cellular proteome forms oligomeric assemblies3. In prokaryotes, a proof-of-concept study showed that assembly of heterodimeric luciferase is an organized cotranslational process, facilitated by spatially confined translation of the subunits encoded on a polycistronic mRNA4. In eukaryotes, however, fundamental differences such as rarity of polycistronic mRNAs and different chaperone constellations raise the question whether assembly is also coordinated with translation. Here we provide a systematic and mechanistic analysis of protein complex assembly in eukaryotes using ribosome profiling. We determined the in vivo nascent subunits interactions of 12 hetero-oligomeric protein complexes of Saccharomyces cerevisiae at near-residue resolution. We find 9 complexes assemble cotranslationally; the 3 complexes that do not show cotranslational interactions are regulated by dedicated assembly chaperones5-7. Cotranslational assembly often occurs uni-directionally, with one fully synthesized subunit engaging its nascent partner subunit(s), thereby counteracting its aggregation propensity. The onset of cotranslational subunit association coincides sharply with full exposure of the nascent interaction domain at the ribosomal tunnel exit. The action of the ribosome-associated Hsp70 chaperone Ssb8 is coordinated with assembly. Ssb transiently engages partially synthesized interaction domains, then dissociates before the onset of partner subunit association, presumably to prevent premature assembly interactions. Our study shows that cotranslational subunit association is a prevalent mechanism for hetero-oligomers assembly in yeast and indicates that translation, folding and assembly of protein complexes are integrated processes in eukaryotes.
2018-07-05 | GSE116570 | GEO
Project description:To understand the orientation of Hsp70 Ssb on the ribosome in living Saccharomyces cerevisiae cells we incorporated the noncanonical, photoactivatable amino acid p-benzoyl-L-phenylalanine (Bpa) in place of specific individual endogenous amino acids in Ssb1. To identify the proteins to which Ssb1Bpa crosslinked we performed mass spectrometry. The results revealed that Bpa incorporated at the tip of the “lid” subdomain cross linked to the nascent chain associated complex (NAC), while Bpa incorporated into further down the lid crosslinked to ribosomal protein uL29.
2024-01-08 | PXD046895 | Pride
Project description:The cellular environment is critical for efficient protein maturation, but how proteins fold during biogenesis remains poorly understood. We used hydrogen/deuterium exchange (HDX) mass spectrometry (MS) to define, at peptide resolution, the cotranslational chaperone-assisted folding pathway of Escherichia coli dihydrofolate reductase. On the ribosome, the nascent polypeptide folds via structured intermediates not populated during refolding from denaturant. Association with the ribosome allows these intermediates to form, as otherwise destabilizing C-terminal sequences remain confined in the ribosome exit tunnel. We find that partially-folded nascent chains recruit the chaperone Trigger factor, which uses a large composite hydrophobic/hydrophilic interface to engage folding intermediates without disrupting their structure. In addition, we comprehensively mapped dynamic interactions between the nascent chain and ribosomal proteins, tracing the path of the emerging polypeptide during synthesis. Our work provides a high-resolution description of de novo protein folding dynamics, thereby revealing new mechanisms by which cellular factors shape the conformational search for the native state.
2024-07-12 | PXD036945 | Pride
Project description:A number of enzymes, targeting factors and chaperones engage ribosomes to support fundamental steps of nascent protein maturation, including enzymatic processing, membrane targeting and co-translational folding. The selective ribosome profiling (SeRP) method is a new tool for studying the co-translational activity of maturation factors that provides proteome-wide information on a factor’s nascent interactome, the onset and duration of binding and the mechanisms controlling factor engagement. SeRP is based on the combination of two ribosome-profiling (RP) experiments, sequencing the ribosome-protected mRNA fragments from all ribosomes (total translatome) and the ribosome subpopulation engaged by the factor of interest (factor-bound translatome). We provide a detailed SeRP protocol, exemplified for the yeast Hsp70 chaperone Ssb (stress 70 B), for studying factor interactions with nascent proteins that is readily adaptable to identifying nascent interactomes of other co-translationally acting eukaryotic factors. The protocol provides general guidance for experimental design and optimization, as well as detailed instructions for cell growth and harvest, the isolation of (factor-engaged) monosomes, the generation of a cDNA library and data analysis. Experience in biochemistry and RNA handling, as well as basic programing knowledge, is necessary to perform SeRP. Execution of a SeRP experiment takes 8–10 working days, and initial data analysis can be completed within 1–2 d. This protocol is an extension of the originally developed protocol describing SeRP in bacteria.
2019-07-10 | GSE123166 | GEO
Project description:Accurate prediction of cellular co-translational folding indicates proteins can switch from post- to co-translational folding
| PRJNA303186 | ENA
Project description:Genome-wide measurement of RNA folding energies
| PRJNA171391 | ENA