Ribosome profiling reveals multiple roles of SecA in cotranslational protein export
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ABSTRACT:
PROVIDER: PRJNA769692 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR16249166.fastq.gz | Fastqsanger.gz | |||
| SRR16249167.fastq.gz | Fastqsanger.gz | |||
| SRR16249168.fastq.gz | Fastqsanger.gz | |||
| SRR16249169.fastq.gz | Fastqsanger.gz | |||
| SRR16249170.fastq.gz | Fastqsanger.gz |
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Project description:SecA, an ATPase known to posttranslationally translocate secretory proteins across the bacterial plasma membrane, also interacts with ribosomes. Here, we used a combination of ribosome profiling methods to investigate the cotranslational actions of SecA in vivo. SecA scans translating ribosomes at the plasma membrane and cotranslationally engages large periplasmic loops on inner membrane proteins. In coordination with the proton motive force, SecA resolves the cytoplasmic accumulation of periplasmic loops during cotranslational protein translocation. SecA also associates with a subset of secretory proteins at an early stage of translation and mediates their cotranslational transport, whereas the chaperone trigger factor (TF) delays SecA engagement on other secretory proteins to impose a posttranslational mode of translocation. The hydrophobicity of signal sequence is a determinant of nascent protein triage between SecA and TF. Our results elucidate the principles of SecA-driven cotranslational protein translocation and reveal a hierarchical network of protein export pathways in bacteria.
2022-05-16 | GSE185572 | GEO
Project description:SecA, an ATPase known to posttranslationally translocate secretory proteins across the bacterial plasma membrane, also binds ribosomes, but the role of SecA's ribosome interaction has been unclear. Here, we used a combination of ribosome profiling methods to investigate the cotranslational actions of SecA. Our data reveal the widespread accumulation of large periplasmic loops of inner membrane proteins in the cytoplasm during their cotranslational translocation, which are specifically recognized and resolved by SecA in coordination with the proton motive force (PMF). Furthermore, SecA associates with 25% of secretory proteins with highly hydrophobic signal sequences at an early stage of translation and mediates their cotranslational transport. In contrast, the chaperone trigger factor (TF) delays SecA engagement on secretory proteins with weakly hydrophobic signal sequences, thus enforcing a posttranslational mode of their translocation. Our results elucidate the principles of SecA-driven cotranslational protein translocation and reveal a hierarchical network of protein export pathways in bacteria.
| S-EPMC9192764 | biostudies-literature
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:The biogenesis of nearly all mitochondrial proteins begins with translation on cytosolic ribosomes. How these proteins are subsequently delivered to mitochondria remains poorly understood. Here, we systematically investigated the coupling of mitochondrial protein translation and import using selective ribosome profiling in human cells. Cotranslational targeting requires an N-terminal presequence on the nascent protein and contributes to mRNA localization at the mitochondrial surface. This pathway is predominantly used by large, multidomain and topologically complex proteins, whose import efficiency is enhanced when targeted cotranslationally. In contrast to protein targeting to the endoplasmic reticulum (ER), cotranslational mitochondrial import does not favor membrane proteins and initiates late during translation, specifically upon the exposure of a complex globular fold in the nascent protein. Our findings reveal a multi-layered protein sorting system that recognizes both the targeting signal and protein folding status during translation.
2025-08-01 | GSE255657 | GEO
Project description:Cotranslational assembly of protein complexes in eukaryotes revealed by ribosome profiling
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Project description:Principles of cotranslational mitochondrial protein import revealed by selective ribosome profiling
| PRJNA1076084 | ENA
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:Cotranslational targeting into the endoplasmic reticulum (ER) by the Signal Recognition Particle (SRP) is a key event determining polypeptide fate in eukaryotic cells. Here, we globally define the principles and mechanisms of SRP binding and ER targeting in vivo. Cotranslational targeting through SRP is the dominant route into the ER for all secretory proteins, regardless of targeting signal characteristics. Cytosolic SRP functions in a pioneer translation round that builds a membrane-resident mRNAs pool, explaining how low SRP levels suffice for the secretory load. SRP does not induce an elongation arrest; consequently, kinetic competition between targeting and translation elongation dictates which substrates are translocated post-translationally. Unexpectedly, SRP binds most secretory ribosomal complexes before targeting signals are synthesized. We show non-coding mRNA elements can promote signal-independent SRP pre-recruitment. Our study defines the complex kinetic interplay between elongation and determinants in the polypeptide and mRNA modulating SRP-substrate selection and membrane targeting in vivo. Ribosome profiling (RiboSeq) and RNA-seq of subcellular fractions of ribosomes. Soluble and membrane bound ribosomes are separated by centrifugation, and SRP-bound ribosomes are immunoprecipitated from the soluble fraction. Polysomes and monosomes are separated by sucrose gradient ultracentrifugation.
2016-08-03 | E-GEOD-74393 | biostudies-arrayexpress
Project description:Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally-distinct classes of chaperone promote de novo folding, suggesting that their activities are coordinated at the ribosome. We used biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We found that chaperone binding is disfavoured close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor recognises compact folding intermediates exposing extensive unfolded surface and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates, and recruits DnaK to sequence-diverse solvent-accessible sites. Neither Trigger factor, DnaJ nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to protect incipient structure in the nascent polypeptide well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates.
2024-07-03 | PXD048623 | Pride
Project description:Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally-distinct classes of chaperones promote de novo folding, suggesting that their activity is coordinated at the ribosome. Here we use biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We find that chaperone binding is disfavoured close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor subsequently recognises compact folding intermediates exposing extensive non-native surface and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates, and recruits DnaK to solvent-accessible sites in a sequence non-specific manner. Neither Trigger factor, DnaJ nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to create a protected space for protein maturation that extends well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates.
2024-07-03 | PXD048645 | Pride