Systematic Localization of <named-content content-type="genus-species">Escherichia coli</named-content> Membrane Proteins

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 physi...

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Autores principales:	Anna Sueki, Frank Stein, Mikhail M. Savitski, Joel Selkrig, Athanasios Typas
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/a7818cd051ee4cd7b901a1f4e66dd29d
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spelling	oai:doaj.org-article:a7818cd051ee4cd7b901a1f4e66dd29d2021-12-02T19:47:38ZSystematic Localization of <named-content content-type="genus-species">Escherichia coli</named-content> Membrane Proteins10.1128/mSystems.00808-192379-5077https://doaj.org/article/a7818cd051ee4cd7b901a1f4e66dd29d2020-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00808-19https://doaj.org/toc/2379-5077ABSTRACT 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.Anna SuekiFrank SteinMikhail M. SavitskiJoel SelkrigAthanasios TypasAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmSystems, Vol 5, Iss 2 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Anna Sueki Frank Stein Mikhail M. Savitski Joel Selkrig Athanasios Typas Systematic Localization of <named-content content-type="genus-species">Escherichia coli</named-content> Membrane Proteins
description	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.
format	article
author	Anna Sueki Frank Stein Mikhail M. Savitski Joel Selkrig Athanasios Typas
author_facet	Anna Sueki Frank Stein Mikhail M. Savitski Joel Selkrig Athanasios Typas
author_sort	Anna Sueki
title	Systematic Localization of <named-content content-type="genus-species">Escherichia coli</named-content> Membrane Proteins
title_short	Systematic Localization of <named-content content-type="genus-species">Escherichia coli</named-content> Membrane Proteins
title_full	Systematic Localization of <named-content content-type="genus-species">Escherichia coli</named-content> Membrane Proteins
title_fullStr	Systematic Localization of <named-content content-type="genus-species">Escherichia coli</named-content> Membrane Proteins
title_full_unstemmed	Systematic Localization of <named-content content-type="genus-species">Escherichia coli</named-content> Membrane Proteins
title_sort	systematic localization of <named-content content-type="genus-species">escherichia coli</named-content> membrane proteins
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/a7818cd051ee4cd7b901a1f4e66dd29d
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Systematic Localization of <named-content content-type="genus-species">Escherichia coli</named-content> Membrane Proteins

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