Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Fluman N

PROVIDER: S-EPMC4359368 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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

Fluman Nir N Navon Sivan S Bibi Eitan E Pilpel Yitzhak Y

eLife 20140818

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 ser ...[more]

PMID: 25135940

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Project description: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.

Dataset Information

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

Publications

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

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