The E. coli Signal Recognition Particle Is Required for the Insertion of a Subset of Inner Membrane Proteins

Recent evidence suggests that in Escherichia coli, SecA/SecB and signal recognition particle (SRP) are constituents of two different pathways targeting secretory and inner membrane proteins to the SecYEG translocon of the plasma membrane. We now show that a secY mutation, which compromises a functional SecY–SecA interaction, does not impair the SRP-mediated integration of polytopic inner membrane proteins. Furthermore, under conditions in which the translocation of secretory proteins is strictly dependent on SecG for assisting SecA, the absence of SecG still allows polytopic membrane proteins to integrate at the wild-type level. These results indicate that SRP-dependent integration and SecA/SecB-mediated translocation do not only represent two independent protein delivery systems, but also remain mechanistically distinct processes even at the level of the membrane where they engage different domains of SecY and different components of the translocon. In addition, the experimental setup used here enabled us to demonstrate that SRP-dependent integration of a multispanning protein into membrane vesicles leads to a biologically active enzyme.

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In Vitro Studies with Purified Components Reveal Signal Recognition Particle (SRP) and SecA/SecB as Constituents of Two Independent Protein-targeting Pathways of Escherichia coli

Molecular Biology of the Cell ◽

10.1091/mbc.10.7.2163 ◽

1999 ◽

Vol 10 (7) ◽

pp. 2163-2173 ◽

Cited By ~ 109

Author(s):

Hans-Georg Koch ◽

Thomas Hengelage ◽

Christoph Neumann-Haefelin ◽

Juan MacFarlane ◽

Hedda K. Hoffschulte ◽

...

Keyword(s):

Escherichia Coli ◽

Plasma Membrane ◽

Membrane Proteins ◽

Signal Recognition Particle ◽

Membrane Vesicles ◽

Secretory Proteins ◽

Signal Recognition ◽

Free System ◽

E Coli

The molecular requirements for the translocation of secretory proteins across, and the integration of membrane proteins into, the plasma membrane of Escherichia coli were compared. This was achieved in a novel cell-free system from E. coliwhich, by extensive subfractionation, was simultaneously rendered deficient in SecA/SecB and the signal recognition particle (SRP) components, Ffh (P48), 4.5S RNA, and FtsY. The integration of two membrane proteins into inside-out plasma membrane vesicles of E. coli required all three SRP components and could not be driven by SecA, SecB, and ΔμH+. In contrast, these were the only components required for the translocation of secretory proteins into membrane vesicles, a process in which the SRP components were completely inactive. Our results, while confirming previous in vivo studies, provide the first in vitro evidence for the dependence of the integration of polytopic inner membrane proteins on SRP in E. coli. Furthermore, they suggest that SRP and SecA/SecB have different substrate specificities resulting in two separate targeting mechanisms for membrane and secretory proteins in E. coli. Both targeting pathways intersect at the translocation pore because they are equally affected by a blocked translocation channel.

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Compensating complete loss of signal recognition particle during co-translational protein targeting by the translation speed and accuracy

10.1101/2021.01.06.425660 ◽

2021 ◽

Author(s):

Liuqun Zhao ◽

Gang Fu ◽

Yanyan Cui ◽

Zixiang Xu ◽

Dawei Zhang

Keyword(s):

Membrane Proteins ◽

Protein Targeting ◽

Time Window ◽

Signal Recognition Particle ◽

Protein Translation ◽

Suppressor Mutation ◽

Signal Recognition ◽

Translation Rate ◽

Inner Membrane ◽

Speed And Accuracy

Signal recognition particle (SRP) is critical for delivering co-translational proteins to the bacterial inner membrane. Previously, we identified SRP suppressors in Escherichia coli that inhibit translation initiation and elongation, which provides an insight into the mechanism of bypassing the requirement of SRP. Suppressor mutations tend to be located in regions that govern protein translation under evolutionary pressure. To verify this hypothesis, we re-executed the suppressor screening of SRP. Here we isolated a novel SRP suppressor mutation located in the Shine-Dalgarno sequence of S10 operon, which partially offset the targeting defects of SRP-dependent proteins. We found that the suppressor mutation slowed the translation rate of proteins, especially of inner membrane proteins, which could compensate for the targeting defects of inner membrane proteins via extending the time window of protein targeting. Furthermore, the fidelity of translation was decreased in suppressor cells, suggesting that the quality control of translation was inactivated to provide a survival advantage under the loss of SRP stress. Our results demonstrate that the inefficient protein targeting due to SRP deletion can be rescued through modulating translational speed and accuracy.

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The Structure of Multiple Polypeptide Domains Determines the Signal Recognition Particle Targeting Requirement ofEscherichia coli Inner Membrane Proteins

Journal of Bacteriology ◽

10.1128/jb.181.15.4561-4567.1999 ◽

1999 ◽

Vol 181 (15) ◽

pp. 4561-4567 ◽

Cited By ~ 20

Author(s):

John A. Newitt ◽

Nancy D. Ulbrandt ◽

Harris D. Bernstein

Keyword(s):

Escherichia Coli ◽

Alkaline Phosphatase ◽

Membrane Proteins ◽

Signal Recognition Particle ◽

Protein Export ◽

Signal Recognition ◽

Inner Membrane ◽

Periplasmic Domain ◽

Membrane Spanning ◽

Membrane Spanning Domains

ABSTRACT The signal recognition particle (SRP) targeting pathway is required for the efficient insertion of many polytopic inner membrane proteins (IMPs) into the Escherichia coli inner membrane, but in the absence of SRP protein export proceeds normally. To define the properties of IMPs that impose SRP dependence, we analyzed the targeting requirements of bitopic IMPs that are structurally intermediate between exported proteins and polytopic IMPs. We found that disruption of the SRP pathway inhibited the insertion of only a subset of bitopic IMPs. Studies on a model bitopic AcrB-alkaline phosphatase fusion protein (AcrB 265-AP) showed that the SRP requirement for efficient insertion correlated with the presence of a large periplasmic domain (P1). As previously reported, perturbation of the SRP pathway also affected the insertion of a polytopic AcrB-AP fusion. Even exhaustive SRP depletion, however, failed to block the insertion of any AcrB derivative by more than 50%. Taken together, these data suggest that many proteins that are normally targeted by SRP can utilize alternative targeting pathways and that the structure of both hydrophilic and membrane-spanning domains determines the degree to which the biogenesis of a protein is SRP dependent.

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Signal recognition particle arrests elongation of nascent secretory and membrane proteins at multiple sites in a transient manner.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)75691-9 ◽

1987 ◽

Vol 262 (4) ◽

pp. 1680-1684

Author(s):

J Lipp ◽

B Dobberstein ◽

M T Haeuptle

Keyword(s):

Membrane Proteins ◽

Signal Recognition Particle ◽

Signal Recognition

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Mechanisms of membrane protein biogenesis

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s205327331408838x ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1161-C1161

Author(s):

Irmgard Sinning

Keyword(s):

Membrane Proteins ◽

Signal Sequence ◽

Signal Recognition Particle ◽

Signal Recognition ◽

Protein Biogenesis ◽

Cargo Proteins ◽

Target Membrane ◽

Hydrophobic Nature ◽

Correct Target ◽

Srp Rna

More than 25% of the cellular proteome comprise membrane proteins that have to be inserted into the correct target membrane. Most membrane proteins are delivered to the membrane by the signal recognition particle (SRP) pathway which relies on the recognition of an N-terminal signal sequence. In contrast to this co-translational mechanism, which avoids problems due to the hydrophobic nature of the cargo proteins, tail-anchored (TA) membrane proteins utilize a post-translational mechanism for membrane insertion – the GET pathway (guided entry of tail-anchored membrane proteins). The SRP and GET pathways are both regulated by GTP and ATP binding proteins of the SIMIBI family. However, in the SRP pathway the SRP RNA plays a unique regulatory role. Recent insights into eukaryotic SRP will be discussed.

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Early encounters of a nascent membrane protein

The Journal of Cell Biology ◽

10.1083/jcb.200503035 ◽

2005 ◽

Vol 170 (1) ◽

pp. 27-35 ◽

Cited By ~ 41

Author(s):

Edith N.G. Houben ◽

Raz Zarivach ◽

Bauke Oudega ◽

Joen Luirink

Keyword(s):

Membrane Protein ◽

Ribosomal Proteins ◽

Transmembrane Domain ◽

Signal Recognition Particle ◽

Chain Complex ◽

Signal Recognition ◽

Inner Membrane ◽

Ribosomal Tunnel ◽

Nascent Chain ◽

Inner Membrane Protein

An unbiased photo–cross-linking approach was used to probe the “molecular path” of a growing nascent Escherichia coli inner membrane protein (IMP) from the peptidyl transferase center to the surface of the ribosome. The nascent chain was initially in proximity to the ribosomal proteins L4 and L22 and subsequently contacted L23, which is indicative of progression through the ribosome via the main ribosomal tunnel. The signal recognition particle (SRP) started to interact with the nascent IMP and to target the ribosome–nascent chain complex to the Sec–YidC complex in the inner membrane when maximally half of the transmembrane domain (TM) was exposed from the ribosomal exit. The combined data suggest a flexible tunnel that may accommodate partially folded nascent proteins and parts of the SRP and SecY. Intraribosomal contacts of the nascent chain were not influenced by the presence of a functional TM in the ribosome.

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Cell-free expression profiling of E. coli inner membrane proteins

PROTEOMICS ◽

10.1002/pmic.200900485 ◽

2010 ◽

Vol 10 (9) ◽

pp. 1762-1779 ◽

Cited By ~ 29

Author(s):

Daniel Schwarz ◽

Daniel Daley ◽

Tobias Beckhaus ◽

Volker Dötsch ◽

Frank Bernhard

Keyword(s):

Membrane Proteins ◽

Expression Profiling ◽

Free Expression ◽

Inner Membrane ◽

E Coli

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Heat Shock Transcription Factor σ32 Co-opts the Signal Recognition Particle to Regulate Protein Homeostasis in E. coli

PLoS Biology ◽

10.1371/journal.pbio.1001735 ◽

2013 ◽

Vol 11 (12) ◽

pp. e1001735 ◽

Cited By ~ 44

Author(s):

Bentley Lim ◽

Ryoji Miyazaki ◽

Saskia Neher ◽

Deborah A. Siegele ◽

Koreaki Ito ◽

...

Keyword(s):

Transcription Factor ◽

Heat Shock ◽

Signal Recognition Particle ◽

Heat Shock Transcription Factor ◽

Protein Homeostasis ◽

Signal Recognition ◽

E Coli ◽

Regulate Protein

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The complexity of pathways for protein import into thylakoids: it's not easy being green

Biochemical Society Transactions ◽

10.1042/bst0331024 ◽

2005 ◽

Vol 33 (5) ◽

pp. 1024-1027 ◽

Cited By ~ 9

Author(s):

A. Di Cola ◽

E. Klostermann ◽

C. Robinson

Keyword(s):

Membrane Proteins ◽

Thylakoid Membrane ◽

Protein Import ◽

Signal Recognition Particle ◽

Integral Membrane Proteins ◽

The Other ◽

Signal Recognition ◽

Transport Mechanisms ◽

Insertion Mechanism

Numerous proteins are transported into or across the chloroplast thylakoid membrane. To date, two major pathways have been identified for the transport of luminal proteins (the Sec- and Tat-dependent pathways) and it is now clear that these protein translocases use fundamentally different transport mechanisms. Integral membrane proteins are inserted by means of at least two further pathways. One involves the input of numerous targeting factors, including SRP (signal recognition particle), FtsY and Albino3. Surprisingly, the other pathway does not involve any of the known chloroplastic targeting factors, and insertion is energy-independent, raising the possibility of an unusual ‘spontaneous’ insertion mechanism.

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