Principles of ER Co-Translational Translocation Revealed by Proximity-Specific Ribosome Profiling
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA260073 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR1562890.fastq.gz | Fastqsanger.gz | |||
| SRR1562900.fastq.gz | Fastqsanger.gz | |||
| SRR1562910.fastq.gz | Fastqsanger.gz | |||
| SRR1562920.fastq.gz | Fastqsanger.gz | |||
| SRR1562930.fastq.gz | Fastqsanger.gz |
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Project description:Localized protein synthesis is a fundamental mechanism for creating distinct subcellular environments. Here we developed a generalizable proximity-specific ribosome profiling strategy that enables global interrogation of translation in defined subcellular locations. We applied this approach to the endoplasmic reticulum (ER) in yeast and mammals. We observed the vast majority of secretory proteins to be co-translationally translocated, including substrates capable of post-translational insertion in vitro. Distinct translocon complexes engaged nascent chains at different points during synthesis. Whereas most proteins engaged the ER immediately following or even before signal sequence (SS) emergence, a class of Sec66-dependent proteins entered with a looped SS conformation. Finally, we observed rapid ribosome exchange into the cytosol following translation termination. These data provide insights into how distinct translocation mechanisms act in concert to promote efficient co-translational recruitment.
2014-11-10 | GSE61012 | GEO
Project description:Localized protein synthesis is a fundamental mechanism for creating distinct subcellular environments. Here we developed a generalizable proximity-specific ribosome profiling strategy that enables global analysis of translation in defined subcellular locations. We applied this approach to the endoplasmic reticulum (ER) in yeast and mammals. We observed the large majority of secretory proteins to be cotranslationally translocated, including substrates capable of posttranslational insertion in vitro. Distinct translocon complexes engaged nascent chains at different points during synthesis. Whereas most proteins engaged the ER immediately after or even before signal sequence (SS) emergence, a class of Sec66-dependent proteins entered with a looped SS conformation. Finally, we observed rapid ribosome exchange into the cytosol after translation termination. These data provide insights into how distinct translocation mechanisms act in concert to promote efficient cotranslational recruitment.
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Project description:Translocon clogging at the endoplasmic reticulum (ER) as a result of translation stalling triggers ribosome UFMylation, activating Translocation-Associated Quality Control (TAQC) to degrade clogged substrates. How cells sense ribosome UFMylation to initiate TAQC is unclear. We conduct a genome-wide CRISPR/Cas9 screen to identify an uncharacterized membrane protein named SAYSD1 that facilitates TAQC. SAYSD1 associates with the Sec61 translocon, and also recognizes both ribosome and UFM1 directly, engaging a stalled nascent chain to ensure its transport via the TRAPP complex to lysosomes for degradation. Like UFM1 deficiency, SAYSD1 depletion causes the accumulation of translocation-stalled proteins at the ER and triggers ER stress. Importantly, disrupting UFM1- and SAYSD1-dependent TAQC in Drosophila leads to intracellular accumulation of translocation-stalled collagens, defective collagen deposition, abnormal basement membranes, and reduced stress tolerance. Thus, SAYSD1 acts as a UFM1 sensor that collaborates with ribosome UFMylation at the site of clogged translocon, safeguarding ER homeostasis during animal development.
2023-01-20 | GSE222104 | GEO
Project description:Nearly all mitochondrial proteins are nuclear-encoded and are targeted to their mitochondrial destination from the cytosol. Here, we used proximity-specific ribosome profiling to comprehensively measure translation at the mitochondrial surface in yeast. The majority of inner membrane proteins were co-translationally targeted to mitochondria, reminiscent of proteins entering the endoplasmic reticulum (ER). Comparison between mitochondrial and ER localization demonstrated that the vast majority of proteins were targeted to a specific organelle. A prominent exception was the fumarate reductase Osm1, known to reside in mitochondria. We identified a conserved ER isoform of Osm1, which contributes to the oxidative protein folding capacity of the organelle. This dual localization was enabled by alternative translation initiation sites encoding distinct targeting signals. These findings highlight the exquisite in vivo specificity of organellar targeting mechanisms.
2014-11-10 | GSE61011 | GEO
Project description:The dynamic ribosome-translocon complex, which resides at the endoplasmic reticulum (ER) membrane, produces a major fraction of the human proteome . It governs the synthesis, translocation, membrane insertion, N-glycosylation, folding and disulfide-bond formation of nascent proteins. While individual components of this machine have been studied at high resolution in isolation, insights into their interplay in the native membrane remain limited. Here, we use electron cryo-tomography (cryo-ET), extensive classification and molecular modeling to capture molecular resolution snapshots of mRNA translation and protein maturation at the ER membrane. Comprehensively recapitulating the translational elongation cycle, we identify a highly abundant classical pre-translocation (PRE) intermediate with eEF1a in an extended conformation following the decoding state, which indicates that eEF1a remains ribosome-associated after GTP-hydrolysis during proofreading. The complete atomic structure of the most abundant ER translocon variant comprising the protein-conducting channel Sec61, the translocon-associated protein complex (TRAP) and the oligosaccharyltransferase complex A (OSTA) reveals the molecular framework for signal peptide (SP) membrane insertion and protein N-glycosylation. Associated with OSTA we observe stoichiometric and sub-stoichiometric cofactors, likely including protein isomerases. Collectively, comprehensive analysis of ER-associated protein biogenesis ex vivo reveals numerous mechanistic insights advancing beyond the study of isolated components.
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Project description:Time-Resolved Photocatalytic Proximity Labeling Uncovers ER Proteome Dynamics Underlying UPR to Apoptosis Transition [RNAseq_EMC2_knockdown]
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Project description:This project was evaluating the orthogonality of proximity labeling peroxidases that are used to determine partitioning of proteins between the ER and the cytosol.
2022-05-08 | PXD033764 | Pride
Project description:The metazoan Sec61 translocon transports polypeptides into and across the membrane of the endoplasmic reticulum via two major routes, a well-established co-translational pathway and a post-translational alternative. We have used two model substrates to explore the elements of a secretory protein precursor that preferentially direct it towards a co- or post-translational pathway for ER translocation. Having first determined the capacity of precursors to enter ER derived microsomes post-translationally, we then exploited semi-permeabilized mammalian cells specifically depleted of key membrane components using siRNA to address their contribution to the membrane translocation process. These studies suggest precursor chain length is a key factor in the post-translational translocation at the mammalian ER, and identify Sec62 and Sec63 as important components acting on this route. This role for Sec62 and Sec63 is independent of the signal sequence that delivers the precursor to the ER. However, the signal sequence can influence the subsequent membrane translocation process, conferring sensitivity to a small molecule inhibitor and dictating reliance on the molecular chaperone BiP. Our data support a model where secretory protein precursors that fail to engage the signal recognition particle, for example because they are short, are delivered to the ER membrane via a distinct route that is dependent upon both Sec62 and Sec63. Although this requirement for Sec62 and Sec63 is unaffected by the specific signal sequence that delivers a precursor to the ER, this region can influence subsequent events, including both Sec61 mediated transport and the importance of BiP for membrane translocation. Taken together, our data suggest that an ER signal sequence can regulate specific aspects of Sec61 mediated membrane translocation at a stage following Sec62/Sec63 dependent ER delivery.
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Project description:In order to elucidate the mechanism underlying ER control of liver metabolic function, we investigated the genetic programs controlled by the hepatic receptor during the physiological fluctuation of circulating E2 in adult female mice. To this aim, we used Affymetrix GeneChip Arrays and compared the expression of hepatic genes in mice in two phases of the estrous cycle: Proestrus (P) and Metestrus (M), characterized by high and low circulating E2 respectively. Analysis of data demonstrated that the expression of genes involved in lipid and cholesterol metabolism changes during the reproductive cycle. To further elucidate ER transcriptional activity at M and P, we performed a whole genome ChIP experiment followed by tiling array. We observed that at M, but not at P, recruited ERs are mostly in proximity (20 kbp) of genes involved in energy metabolism, thus strengthening the hypothesis of ER playing a bridging role between metabolism and reproduction.
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