Structure of the Mammalian Ribosome-Sec61 Complex to 3.4 Å Resolution

Voorhees, R.M.; Fernandez, I.S.; Scheres, Sjors H.W.; Hegde, Ramanujan S.

doi:10.1016/j.cell.2014.05.024

Cited by 318 publications

(446 citation statements)

References 72 publications

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“…A critical role for Sec61 in protein export to the cytosol is also not fully consistent with the pore size that is required to support the export of functional fully folded proteins, such as β-lactamase or HRP, or heavily glycosylated proteins, such as OVA (31). If Sec61 is not involved, how then do antigens escape endocytic compartments in DCs?…”

Section: Discussionmentioning

confidence: 99%

Sec61 blockade by mycolactone inhibits antigen cross-presentation independently of endosome-to-cytosol export

Grotzke

Kozik

Morel

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Although antigen cross-presentation in dendritic cells (DCs) is critical to the initiation of most cytotoxic immune responses, the intracellular mechanisms and traffic pathways involved are still unclear. One of the most critical steps in this process, the export of internalized antigen to the cytosol, has been suggested to be mediated by Sec61. Sec61 is the channel that translocates signal peptide-bearing nascent polypeptides into the endoplasmic reticulum (ER), and it was also proposed to mediate protein retrotranslocation during ER-associated degradation (a process called ERAD). Here, we used a newly identified Sec61 blocker, mycolactone, to analyze Sec61's contribution to antigen cross-presentation, ERAD, and transport of internalized antigens into the cytosol. As shown previously in other cell types, mycolactone prevented protein import into the ER of DCs. Mycolactone-mediated Sec61 blockade also potently suppressed both antigen cross-presentation and direct presentation of synthetic peptides to CD8 + T cells. In contrast, it did not affect protein export from the ER lumen or from endosomes into the cytosol, suggesting that the inhibition of cross-presentation was not related to either of these trafficking pathways. Proteomic profiling of mycolactoneexposed DCs showed that expression of mediators of antigen presentation, including MHC class I and β2 microglobulin, were highly susceptible to mycolactone treatment, indicating that Sec61 blockade affects antigen cross-presentation indirectly. Together, our data suggest that the defective translocation and subsequent degradation of Sec61 substrates is the cause of altered antigen cross-presentation in Sec61-blocked DCs.Sec61 | cross-presentation | ERAD | mycolactone

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Section: Discussionmentioning

confidence: 99%

Sec61 blockade by mycolactone inhibits antigen cross-presentation independently of endosome-to-cytosol export

Grotzke

Kozik

Morel

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Accurate selection of the nascent protein into the correct biogenesis pathway is essential to maintain the homeostasis of the proteome. In recent years, there has been an increasing number of structures of individual protein biogenesis factors bound to the ribosome exit site (3,18,26,56,(66)(67)(68)(69)(70)(71)(72)(73)(74), as well as efforts to globally catalog the nascent polypeptides they occupy (9, 13). However, the key question remains: Given the crowded environment at the ribosome exit site and a limited time window of action, how does a nascent polypeptide engage the correct set of factors and hence commit to its correct biogenesis pathway?…”

Section: Discussionmentioning

confidence: 99%

Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting

Ariosa

Lee

Wang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The ribosome exit site is a crowded environment where numerous factors contact nascent polypeptides to influence their folding, localization, and quality control. Timely and accurate selection of nascent polypeptides into the correct pathway is essential for proper protein biogenesis. To understand how this is accomplished, we probe the mechanism by which nascent polypeptides are accurately sorted between the major cotranslational chaperone trigger factor (TF) and the essential cotranslational targeting machinery, signal recognition particle (SRP). We show that TF regulates SRP function at three distinct stages, including binding of the translating ribosome, membrane targeting via recruitment of the SRP receptor, and rejection of ribosome-bound nascent polypeptides beyond a critical length. Together, these mechanisms enhance the specificity of substrate selection into both pathways. Our results reveal a multilayered mechanism of molecular interplay at the ribosome exit site, and provide a conceptual framework to understand how proteins are selected among distinct biogenesis machineries in this crowded environment.signal recognition particle | trigger factor | ribosome | protein biogenesis | GTPases P roper protein biogenesis is a prerequisite for the maintenance of a functional proteome. Accumulating data indicate that this process begins at the ribosome exit site, where many protein biogenesis machineries can interact and gain access to the nascent polypeptide. This includes chaperones (1-5) such as trigger factor (TF) (1, 4, 6, 7), Hsp70, and the nascent polypeptide-associated complex (8-13); modification enzymes (10, 14-16) such as N-acetyl transferase, methionine aminopeptidase, and arginyl transferase; protein-targeting and translocation machineries such as signal recognition particle (SRP) (17-20), SecA (21), the SecYEG (or Sec61p) (22, 23) and YidC translocases (24,25), and the ribosome-bound quality control complex (26)(27)(28)(29)(30). Engagement of these factors with nascent polypeptides influences their folding, assembly, localization, processing, and quality control. Within seconds after the nascent polypeptide emerges from the ribosomal exit tunnel, it must engage the correct set of factors and thus commit to the proper biogenesis pathway. How this is accomplished in the crowded environment at the ribosome exit site is an emerging question. In this work, we address this question by deciphering how nascent proteins are selected between two major protein biogenesis machineries in bacteria, SRP and TF.SRP is a universally conserved ribonucleoprotein complex responsible for the cotranslational targeting of proteins to the eukaryotic endoplasmic reticulum (ER), or the bacterial plasma membrane (31). SRP recognizes ribosome-nascent chain complexes (termed RNC or cargo) carrying strong signal sequences and delivers them to the SecYEG or YidC translocation machinery on the target membrane. SRP binds RNC via two interactions: a helical N domain in the SRP54 protein (called Ffh in bacteria) binds the ribosoma...

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“…The β-hairpin extends deep inside the ribosome so that its tip faces the exit tunnel and forms additional hydrophobic/electrostatic contacts with a hairpin loop in domain III of 28S rRNA ( Fig. 4F; Voorhees et al 2014). The absence of ten 5…”

Section: Rpl17 Deficiency Altersmentioning

confidence: 99%

“…4A). To obtain insight into possible consequences of this disruption, we looked at the local ribosome environment in a highresolution structure of the mammalian Rpl17 affects diversity of mammalian 5.8S rRNA www.rnajournal.org 1245 ribosome-Sec61 complex (Voorhees et al 2014). Helix 2 in a mature 60S subunit is positioned in close proximity to Rpl17, which interacts with 28S and 5.8S rRNAs at several sites (Fig.…”

Section: Rpl17 Deficiency Altersmentioning

confidence: 99%

Reduced expression of the mouse ribosomal protein Rpl17 alters the diversity of mature ribosomes by enhancing production of shortened 5.8S rRNA

Wang

Parshin

Shcherbik

et al. 2015

RNA

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Processing of rRNA during ribosome assembly can proceed through alternative pathways but it is unclear whether this could affect the structure of the ribosome. Here, we demonstrate that shortage of a ribosomal protein can change pre-rRNA processing in a way that over time alters ribosome diversity in the cell. Reducing the amount of Rpl17 in mouse cells led to stalled 60S subunit maturation, causing degradation of most of the synthesized precursors. A fraction of pre-60S subunits, however, were able to complete maturation, but with a 5 ′ -truncated 5.8S rRNA, which we named 5.8S C . The 5 ′ exoribonuclease Xrn2 is involved in the generation of both 5.8S C and the canonical long form of 5.8S rRNA. Ribosomes containing 5.8S C rRNA are present in various mouse and human cells and engage in translation. These findings uncover a previously undescribed form of mammalian 5.8S rRNA and demonstrate that perturbations in ribosome assembly can be a source of heterogeneity in mature ribosomes.

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Structure of the Mammalian Ribosome-Sec61 Complex to 3.4 Å Resolution

Cited by 318 publications

References 72 publications

Sec61 blockade by mycolactone inhibits antigen cross-presentation independently of endosome-to-cytosol export

Sec61 blockade by mycolactone inhibits antigen cross-presentation independently of endosome-to-cytosol export

Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting

Reduced expression of the mouse ribosomal protein Rpl17 alters the diversity of mature ribosomes by enhancing production of shortened 5.8S rRNA

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