mRNA-programmed translation pauses in the targeting of E. coli membrane proteins

In all living organisms, ribosomes translating membrane proteins are targeted to membrane translocons early in translation, by the ubiquitous signal recognition particle (SRP) system. In eukaryotes, the SRP Alu domain arrests translation elongation of membrane proteins until targeting is complete. Curiously, however, the Alu domain is lacking in most eubacteria. In this study, by analyzing genome-wide data on translation rates, we identified a potential compensatory mechanism in E. coli that serves to slow down the translation during membrane protein targeting. The underlying mechanism is likely programmed into the coding sequence, where Shine–Dalgarno-like elements trigger elongation pauses at strategic positions during the early stages of translation. We provide experimental evidence that slow translation during targeting and improves membrane protein production fidelity, as it correlates with better folding of overexpressed membrane proteins. Thus, slow elongation is important for membrane protein targeting in E. coli, which utilizes mechanisms different from the eukaryotic one to control the translation speed.

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Tail-Anchored Inner Membrane Protein ElaB Increases Resistance to Stress While Reducing Persistence in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00057-17 ◽

2017 ◽

Vol 199 (9) ◽

Cited By ~ 11

Author(s):

Yunxue Guo ◽

Xiaoxiao Liu ◽

Baiyuan Li ◽

Jianyun Yao ◽

Thomas K. Wood ◽

...

Keyword(s):

Oxidative Stress ◽

Escherichia Coli ◽

Membrane Proteins ◽

Membrane Protein ◽

Stationary Phase ◽

Transmembrane Domain ◽

Inner Membrane ◽

Content Type ◽

E Coli ◽

Inner Membrane Protein

ABSTRACT Host-associated bacteria, such as Escherichia coli, often encounter various host-related stresses, such as nutritional deprivation, oxidative stress, and temperature shifts. There is growing interest in searching for small endogenous proteins that mediate stress responses. Here, we characterized the small C-tail-anchored inner membrane protein ElaB in E. coli. ElaB belongs to a class of tail-anchored inner membrane proteins with a C-terminal transmembrane domain but lacking an N-terminal signal sequence for membrane targeting. Proteins from this family have been shown to play vital roles, such as in membrane trafficking and apoptosis, in eukaryotes; however, their role in prokaryotes is largely unexplored. Here, we found that the transcription of elaB is induced in the stationary phase in E. coli and stationary-phase sigma factor RpoS regulates elaB transcription by binding to the promoter of elaB. Moreover, ElaB protects cells against oxidative stress and heat shock stress. However, unlike membrane peptide toxins TisB and GhoT, ElaB does not lead to cell death, and the deletion of elaB greatly increases persister cell formation. Therefore, we demonstrate that disruption of C-tail-anchored inner membrane proteins can reduce stress resistance; it can also lead to deleterious effects, such as increased persistence, in E. coli. IMPORTANCE Escherichia coli synthesizes dozens of poorly understood small membrane proteins containing a predicted transmembrane domain. In this study, we characterized the function of the C-tail-anchored inner membrane protein ElaB in E. coli. ElaB increases resistance to oxidative stress and heat stress, while inactivation of ElaB leads to high persister cell formation. We also demonstrated that the transcription of elaB is under the direct regulation of stationary-phase sigma factor RpoS. Thus, our study reveals that small inner membrane proteins may have important cellular roles during the stress response.

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Compensating Complete Loss of Signal Recognition Particle During Co-translational Protein Targeting by the Translation Speed and Accuracy

Frontiers in Microbiology ◽

10.3389/fmicb.2021.690286 ◽

2021 ◽

Vol 12 ◽

Author(s):

Liuqun Zhao ◽

Gang Fu ◽

Yanyan Cui ◽

Zixiang Xu ◽

Tao Cai ◽

...

Keyword(s):

Protein Targeting ◽

Time Window ◽

Signal Recognition Particle ◽

Protein Translation ◽

Suppressor Mutation ◽

Signal Recognition ◽

Translation Rate ◽

Translation Speed ◽

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 provided insights into the mechanism of bypassing the requirement of SRP. Suppressor mutations tended to be located in regions that govern protein translation under evolutionary pressure. To test 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 the S10 operon, which partially offset the targeting defects of SRP-dependent proteins. We found that the suppressor mutation decreased the protein translation rate, which extended the time window of protein targeting. This increased the possibility of the correct localization of inner membrane proteins. Furthermore, the fidelity of translation was decreased in suppressor cells, suggesting that the quality control of translation was inactivated to provide an advantage in tolerating toxicity caused by the loss of SRP. Our results demonstrated that the inefficient protein targeting due to SRP deletion can be rescued through modulating translational speed and accuracy.

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Distinct Requirements for Tail-Anchored Membrane Protein Biogenesis in Escherichia coli

mBio ◽

10.1128/mbio.01580-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 1

Author(s):

Markus Peschke ◽

Mélanie Le Goff ◽

Gregory M. Koningstein ◽

Norbert O. Vischer ◽

Abbi Abdel-Rehim ◽

...

Keyword(s):

Escherichia Coli ◽

Membrane Proteins ◽

Transmembrane Domain ◽

Signal Recognition Particle ◽

Membrane Insertion ◽

Type Ii ◽

Content Type ◽

E Coli ◽

C Terminus

ABSTRACT Tail-anchored membrane proteins (TAMPs) are a distinct subset of inner membrane proteins (IMPs) characterized by a single C-terminal transmembrane domain (TMD) that is responsible for both targeting and anchoring. Little is known about the routing of TAMPs in bacteria. Here, we have investigated the role of TMD hydrophobicity in tail-anchor function in Escherichia coli and its influence on the choice of targeting/insertion pathway. We created a set of synthetic, fluorescent TAMPs that vary in the hydrophobicity of their TMDs and corresponding control polypeptides that are extended at their C terminus to create regular type II IMPs. Surprisingly, we observed that TAMPs have a much lower TMD hydrophobicity threshold for efficient targeting and membrane insertion than their type II counterparts. Using strains conditional for the expression of known membrane-targeting and insertion factors, we show that TAMPs with strongly hydrophobic TMDs require the signal recognition particle (SRP) for targeting. Neither the SecYEG translocon nor YidC appears to be essential for the membrane insertion of any of the TAMPs studied. In contrast, corresponding type II IMPs with a TMD of sufficient hydrophobicity to promote membrane insertion followed an SRP- and SecYEG translocon-dependent pathway. Together, these data indicate that the capacity of a TMD to promote the biogenesis of E. coli IMPs is strongly dependent upon the polypeptide context in which it is presented. IMPORTANCE A subset of membrane proteins is targeted to and inserted into the membrane via a hydrophobic transmembrane domain (TMD) that is positioned at the very C terminus of the protein. The biogenesis of these so-called tail-anchored proteins (TAMPs) has been studied in detail in eukaryotic cells. Various partly redundant pathways were identified, the choice for which depends in part on the hydrophobicity of the TMD. Much less is known about bacterial TAMPs. The significance of our research is in identifying the role of TMD hydrophobicity in the routing of E. coli TAMPs. Our data suggest that both the nature of the TMD and its role in routing can be very different for TAMPs versus “regular” membrane proteins. Elucidating these position-specific effects of TMDs will increase our understanding of how prokaryotic cells face the challenge of producing a wide variety of membrane proteins.

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

Cell ◽

10.1016/s0092-8674(00)81839-5 ◽

1997 ◽

Vol 88 (2) ◽

pp. 187-196 ◽

Cited By ~ 240

Author(s):

Nancy D Ulbrandt ◽

John A Newitt ◽

Harris D Bernstein

Keyword(s):

Membrane Proteins ◽

Signal Recognition Particle ◽

Signal Recognition ◽

Inner Membrane ◽

E Coli

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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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Escherichia coli SRP, Its Protein Subunit Ffh, and the Ffh M Domain Are Able To Selectively Limit Membrane Protein Expression When Overexpressed

mBio ◽

10.1128/mbio.00020-10 ◽

2010 ◽

Vol 1 (2) ◽

Cited By ~ 11

Author(s):

Ido Yosef ◽

Elena S. Bochkareva ◽

Eitan Bibi

Keyword(s):

Escherichia Coli ◽

Quality Control ◽

Membrane Proteins ◽

Membrane Protein ◽

Protein Translation ◽

Mrna Levels ◽

Content Type ◽

E Coli ◽

Quality Control Process

ABSTRACT The Escherichia coli signal recognition particle (SRP) system plays an important role in membrane protein biogenesis. Previous studies have suggested indirectly that in addition to its role during the targeting of ribosomes translating membrane proteins to translocons, the SRP might also have a quality control role in preventing premature synthesis of membrane proteins in the cytoplasm. This proposal was studied here using cells simultaneously overexpressing various membrane proteins and either SRP, the SRP protein Ffh, its 4.5S RNA, or the Ffh M domain. The results show that SRP, Ffh, and the M domain are all able to selectively inhibit the expression of membrane proteins. We observed no apparent changes in the steady-state mRNA levels or membrane protein stability, suggesting that inhibition may occur at the level of translation, possibly through the interaction between Ffh and ribosome-hydrophobic nascent chain complexes. Since E. coli SRP does not have a eukaryote-like translation arrest domain, we discuss other possible mechanisms by which this SRP might regulate membrane protein translation when overexpressed. IMPORTANCE The eukaryotic SRP slows down translation of SRP substrates by cytoplasmic ribosomes. This activity is important for preventing premature synthesis of secretory and membrane proteins in the cytoplasm. It is likely that an analogous quality control step would be required in all living cells. However, on the basis of its composition and domain structure and limited in vitro studies, it is believed that the E. coli SRP is unable to regulate ribosomes translating membrane proteins. Nevertheless, several in vivo studies have suggested otherwise. To address this issue further in vivo, we utilized unbalanced conditions under which E. coli simultaneously overexpresses SRP and each of several membrane or cytosolic proteins. Surprisingly, our results clearly show that the E. coli SRP is capable of regulating membrane protein synthesis and demonstrate that the M domain of Ffh mediates this activity. These results thus open the way for mechanistic characterization of this quality control process in bacteria.

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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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Systematic Localization of Escherichia coli Membrane Proteins

mSystems ◽

10.1128/msystems.00808-19 ◽

2020 ◽

Vol 5 (2) ◽

Cited By ~ 2

Author(s):

Anna Sueki ◽

Frank Stein ◽

Mikhail M. Savitski ◽

Joel Selkrig ◽

Athanasios Typas

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Protein Localization ◽

Sucrose Density Gradient ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Outer Membranes ◽

Protein Databases ◽

Soluble Cell

ABSTRACT The molecular architecture and function of the Gram-negative bacterial cell envelope are dictated by protein composition and localization. Proteins that localize to the inner membranes (IM) and outer membranes (OM) of Gram-negative bacteria play critical and distinct roles in cellular physiology; however, approaches to systematically interrogate their distribution across both membranes and the soluble cell fraction are lacking. Here, we employed multiplexed quantitative mass spectrometry using tandem mass tag (TMT) labeling to assess membrane protein localization in a proteome-wide fashion by separating IM and OM vesicles from exponentially growing Escherichia coli K-12 cells on a sucrose density gradient. The migration patterns for >1,600 proteins were classified in an unbiased manner, accurately recapitulating decades of knowledge in membrane protein localization in E. coli. For 559 proteins that are currently annotated as peripherally associated with the IM (G. Orfanoudaki and A. Economou, Mol Cell Proteomics 13:3674–3687, 2014, https://doi.org/10.1074/mcp.O114.041137) and that display potential for dual localization to either the IM or cytoplasm, we could allocate 110 proteins to the IM and 206 proteins to the soluble cell fraction based on their fractionation patterns. In addition, we uncovered 63 cases, in which our data disagreed with current localization annotation in protein databases. For 42 of these cases, we were able to find supportive evidence for our localization findings in the literature. We anticipate that our systems-level analysis of the E. coli membrane proteome will serve as a useful reference data set to query membrane protein localization, as well as to provide a novel methodology to rapidly and systematically map membrane protein localization in more poorly characterized Gram-negative species. IMPORTANCE Current knowledge of protein localization, particularly outer membrane proteins, is highly dependent on bioinformatic predictions. To date, no systematic experimental studies have directly compared protein localization spanning the inner and outer membranes of E. coli. By combining sucrose density gradient fractionation of inner membrane (IM) and outer membrane (OM) proteins with multiplex quantitative proteomics, we systematically quantified localization patterns for >1,600 proteins, providing high-confidence localization annotations for 1,368 proteins. Of these proteins, we resolve the predominant localization of 316 proteins that currently have dual annotation (cytoplasmic and IM) in protein databases and identify new annotations for 42 additional proteins. Overall, we present a novel quantitative methodology to systematically map membrane proteins in Gram-negative bacteria and use it to unravel the biological complexity of the membrane proteome architecture in E. coli.

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Getting on target: The archaeal signal recognition particle

Archaea ◽

10.1155/2002/729649 ◽

2002 ◽

Vol 1 (1) ◽

pp. 27-34 ◽

Cited By ~ 32

Author(s):

Christian Zwieb ◽

Jerry Eichler

Keyword(s):

Membrane Proteins ◽

Biochemical Analysis ◽

Protein Targeting ◽

Polypeptide Chain ◽

Protein Translocation ◽

Signal Recognition Particle ◽

Evolutionary Significance ◽

Signal Recognition ◽

Functional Behavior ◽

Translocation Pathway

Protein translocation begins with the efficient targeting of secreted and membrane proteins to complexes embedded within the membrane. In Eukarya and Bacteria, this is achieved through the interaction of the signal recognition particle (SRP) with the nascent polypeptide chain. In Archaea, homologs of eukaryal and bacterial SRP-mediated translocation pathway components have been identified. Biochemical analysis has revealed that although the archaeal system incorporates various facets of the eukaryal and bacterial targeting systems, numerous aspects of the archaeal system are unique to this domain of life. Moreover, it is becoming increasingly clear that elucidation of the archaeal SRP pathway will provide answers to basic questions about protein targeting that cannot be obtained from examination of eukaryal or bacterial models. In this review, recent data regarding the molecular composition, functional behavior and evolutionary significance of the archaeal signal recognition particle pathway are discussed.

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