Rapid label‐free quantitative analysis of the <i>E. coli</i> BL21(DE3) inner membrane proteome

Papanastasiou, Malvina; Orfanoudaki, Georgia; Kountourakis, Nikos; Koukaki, Marina; Sardis, Marios Frantzeskos; Aivaliotis, Michalis; Tsolis, Konstantinos C.; Karamanou, Spyridoula; Economou, Anastassios

doi:10.1002/pmic.201500304

Cited by 25 publications

(22 citation statements)

References 91 publications

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“…We were able to explain 51 by additional literature data. Those proteins have a wrong or missing annotation in STEPdb (42) or their function/activity makes their sucrose gradient fractionation patterns misleading (8). In most cases, sucrose gradient fractionation failed to make the right call when the protein was spanning the envelope or presumably had dual membrane localization.…”

Section: Discussionmentioning

confidence: 99%

“…Proteomic-based studies of bacterial cell membranes in the past have focused on either the IM or OM proteomes separately (39)(40)(41)(42), which precludes definitive statements about the protein allocation across the two membranes and is prone to contamination from abundant soluble proteins. To systematically examine membrane protein localization in an unbiased and systematic manner, we combined sucrose gradient membrane fractionation with quantitative proteomics (43).…”

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confidence: 99%

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Systematic Localization of Escherichia coli Membrane Proteins

et al. 2020

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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 Ͼ1,600 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 Ͼ1,600 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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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Systematic Localization of Escherichia coli Membrane Proteins

et al. 2020

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“…By analogy to CsrB/C, GcvB turnover could require interaction with a regulatory factor on the inner membrane. Proteomics surveys have shown that there are over 700 proteins in E. coli that are either directly or indirectly associated with the inner membrane, suggesting the potential for numerous interactions with RNase E in the posttranscriptional control of gene expression (Papanastasiou et al , ; Papanastasiou et al , ).…”

Section: Discussionmentioning

confidence: 99%

Detachment of the RNA degradosome from the inner membrane of Escherichia coli results in a global slowdown of mRNA degradation, proteolysis of RNase E and increased turnover of ribosome‐free transcripts

Hadjeras

Poljak

Bouvier

et al. 2019

Molecular Microbiology

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SummaryThe reason for RNase E attachment to the inner membrane is largely unknown. To understand the cell biology of RNA degradation, we have characterized a strain expressing RNase E lacking the membrane attachment site (cytoplasmic RNase E). Genome‐wide data show a global slowdown in mRNA degradation. There is no correlation between mRNA stabilization and the function or cellular location of encoded proteins. The activity of cRNase E is comparable to the wild‐type enzyme in vitro, but the mutant protein is unstable in vivo. Autoregulation of cRNase E synthesis compensates for protein instability. cRNase E associates with other proteins to assemble a cytoplasmic RNA degradosome. CsrB/C sRNAs, whose stability is regulated by membrane‐associated CsrD, are stabilized. Membrane attachment of RNase E is thus necessary for CsrB/C turnover. In contrast to mRNA stability, ribosome‐free transcripts are sensitive to inactivation by cRNase E. Our results show that effects on RNA degradation are not due to the differences in the activity or level of cRNase E, or failure to assemble the RNA degradosome. We propose that membrane attachment is necessary for RNase E stability, functional interactions with membrane‐associated regulatory factors and protection of ribosome‐free transcripts from premature interactions with RNase E in the nucleoid.

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“…In addition to predicted cell envelope proteins, there were also soluble proteins which are known to associate into macromolecular complexes, such as the ribosome or GroEL complex. Previous reports have shown that these large assemblies are prone to co-sedimenting with the cell membrane fraction during the ultracentrifugation step after cell lysis, thus explaining their presence in our peptidisc library preparations (Papanastasiou et al 2013(Papanastasiou et al , 2015.…”

Section: Fractionation Of the Silac-labeled Peptidisc Librarymentioning

confidence: 71%

Profiling the E. coli Membrane Interactome Captured in Peptidisc Libraries

Carlson

Stacey

Young

et al. 2019

Preprint

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Protein-correlation-profiling (PCP), in combination with quantitative proteomics, has emerged as a high-throughput method for the rapid identification of dynamic protein complexes in native conditions. While PCP has been successfully applied to soluble proteomes, characterization of the membrane interactome has lagged, partly due to the necessary use of detergents to maintain protein solubility. Here, we apply the peptidisc, a 'one-size fits all' membrane mimetic, for the capture of the Escherichia coli cell envelope proteome and its high-resolution fractionation in the absence of detergent. Analysis of the SILAC-labeled peptidisc library via PCP allows generation of over 4900 possible binary interactions out of >700,000 random associations. Using well-characterized membrane protein systems such as the SecY translocon, the Bam complex and the MetNI transporter, we find our dataset very useful for identifying transient and surprisingly novel protein interactions. For example, we discover a trans-periplasmic supercomplex comprising subunits of the Bam and Sec translocons, including membrane-bound chaperones YfgM and PpiD. We identify RcsF and OmpA as bone fide interactors of BamA, and we show that interaction of MetQ with the ABC transporter MetNI depends on its N-terminal lipid anchor. We also discover NlpA as a novel interactor of MetNIQ. Most of these interactions are largely undetected by standard detergent-based purification. Together, the peptidisc workflow applied to the proteomic field is emerging as a promising novel approach to characterize membrane protein interactions under native expression conditions and without genetic manipulation.

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Rapid label‐free quantitative analysis of the E. coli BL21(DE3) inner membrane proteome

Cited by 25 publications

References 91 publications

Systematic Localization of Escherichia coli Membrane Proteins

Systematic Localization of Escherichia coli Membrane Proteins

Detachment of the RNA degradosome from the inner membrane of Escherichia coli results in a global slowdown of mRNA degradation, proteolysis of RNase E and increased turnover of ribosome‐free transcripts

Profiling the E. coli Membrane Interactome Captured in Peptidisc Libraries

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