SecA Interacts with Ribosomes in Order to Facilitate Posttranslational Translocation in Bacteria

Huber, Damon; Rajagopalan, Nandhakishore; Preißler, Steffen; Rocco, Mark A.; Merz, Frieder; Krämer, Günter; Bukau, Bernd

doi:10.1016/j.molcel.2010.12.028

Cited by 99 publications

(160 citation statements)

References 52 publications

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“…4B). This is also consistent with recent studies indicating cross-linking of SecA to L23 protein (38).…”

Section: Resultssupporting

confidence: 94%

“…Almost no binding of SecA was detected when the salt concentration was increased to 500 mM, thus confirming the salt-sensitive, specific binding of SecA to the ribosome (Fig. 1A) proposed recently (38). SecA binding to ribosomes almost showed saturation when a 5-fold molar excess of SecA was used because binding did not increase significantly when the SecA concentration was increased to a 10-fold molar excess (Fig.…”

Section: Resultssupporting

confidence: 87%

“…SecA has been shown to interact with translating ribosomes (37), and, recently, Huber et al (38) suggested that the monomeric form of SecA binds to the ribosomes through L23 protein on the polypeptide tunnel exit.…”

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confidence: 99%

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Cryo-electron Microscopic Structure of SecA Protein Bound to the 70S Ribosome

Singh

Kraft

Jaiswal

et al. 2014

Journal of Biological Chemistry

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“…4B). This is also consistent with recent studies indicating cross-linking of SecA to L23 protein (38).…”

Section: Resultssupporting

confidence: 94%

Section: Resultssupporting

confidence: 87%

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Cryo-electron Microscopic Structure of SecA Protein Bound to the 70S Ribosome

Singh

Kraft

Jaiswal

et al. 2014

Journal of Biological Chemistry

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“…SecAmediated targeting to the bacterial cytoplasmic membrane is considered to be a posttranslational process (6,37), but our results show unambiguously that the PetA signal sequence engages the thylakoid membrane shortly after it emerges from the ribosome. Bacterial SecA is bound to the ribosome (38) and could potentially bind signal sequences cotranslationally. Assays similar to those used here could be used to address whether this in fact occurs.…”

Section: Discussionmentioning

confidence: 99%

Genome-wide analysis of thylakoid-bound ribosomes in maize reveals principles of cotranslational targeting to the thylakoid membrane

Zoschke

Barkan

2015

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

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Chloroplast genomes encode ∼37 proteins that integrate into the thylakoid membrane. The mechanisms that target these proteins to the membrane are largely unexplored. We used ribosome profiling to provide a comprehensive, high-resolution map of ribosome positions on chloroplast mRNAs in separated membrane and soluble fractions in maize seedlings. The results show that translation invariably initiates off the thylakoid membrane and that ribosomes synthesizing a subset of membrane proteins subsequently become attached to the membrane in a nucleaseresistant fashion. The transition from soluble to membraneattached ribosomes occurs shortly after the first transmembrane segment in the nascent peptide has emerged from the ribosome. Membrane proteins whose translation terminates before emergence of a transmembrane segment are translated in the stroma and targeted to the membrane posttranslationally. These results indicate that the first transmembrane segment generally comprises the signal that links ribosomes to thylakoid membranes for cotranslational integration. The sole exception is cytochrome f, whose cleavable N-terminal cpSecA-dependent signal sequence engages the thylakoid membrane cotranslationally. The distinct behavior of ribosomes synthesizing the inner envelope protein CemA indicates that sorting signals for the thylakoid and envelope membranes are distinguished cotranslationally. In addition, the fractionation behavior of ribosomes in polycistronic transcription units encoding both membrane and soluble proteins adds to the evidence that the removal of upstream ORFs by RNA processing is not typically required for the translation of internal genes in polycistronic chloroplast mRNAs.ribosome profiling | protein targeting | plastid | chloroplast | SecA

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“…How are nascent proteins sorted between them and committed to the correct pathway in a timely and accurate manner? Extensive past work to address these questions has led to different (and sometimes contradictory) models, including (i) TF and SRP compete for binding to the RNC (10,15,16,18); (ii) TF and SRP can bind to the same RNC simultaneously (17,(19)(20)(21); (iii) FtsY rejects TF from SRP-bound ribosomes (17); and (iv) TF preferentially occupies longer nascent chains (13,(45)(46)(47) and, by inference, SRP preferentially binds short nascent chains. A unifying model that reconciles all these observations and explains how nascent chains on the ribosome are selected by TF or SRP is still lacking.…”

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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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SecA Interacts with Ribosomes in Order to Facilitate Posttranslational Translocation in Bacteria

Cited by 99 publications

References 52 publications

Cryo-electron Microscopic Structure of SecA Protein Bound to the 70S Ribosome

Cryo-electron Microscopic Structure of SecA Protein Bound to the 70S Ribosome

Genome-wide analysis of thylakoid-bound ribosomes in maize reveals principles of cotranslational targeting to the thylakoid membrane

Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting

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