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Protein localization in Escherichia coli cells: comparison of the cytoplasmic membrane proteins ProP, LacY, ProW, AqpZ, MscS, and MscL.

ABSTRACT: Fluorescence microscopy has revealed that the phospholipid cardiolipin (CL) and FlAsH-labeled transporters ProP and LacY are concentrated at the poles of Escherichia coli cells. The proportion of CL among E. coli phospholipids can be varied in vivo as it is decreased by cls mutations and it increases with the osmolality of the growth medium. In this report we compare the localization of CL, ProP, and LacY with that of other cytoplasmic membrane proteins. The proportion of cells in which FlAsH-labeled membrane proteins were concentrated at the cell poles was determined as a function of protein expression level and CL content. Each tagged protein was expressed from a pBAD24-derived plasmid; tagged ProP was also expressed from the chromosome. The osmosensory transporter ProP and the mechanosensitive channel MscS concentrated at the poles at frequencies correlated with the cellular CL content. The lactose transporter LacY was found at the poles at a high and CL-independent frequency. ProW (a component of the osmoregulatory transporter ProU), AqpZ (an aquaporin), and MscL (a mechanosensitive channel) were concentrated at the poles in a minority of cells, and this polar localization was CL independent. The frequency of polar localization was independent of induction (at arabinose concentrations up to 1 mM) for proteins encoded by pBAD24-derived plasmids. Complementation studies showed that ProW, AqpZ, MscS, and MscL remained functional after introduction of the FlAsH tag (CCPGCC). These data suggest that CL-dependent polar localization in E. coli cells is not a general characteristic of transporters, channels, or osmoregulatory proteins. Polar localization can be frequent and CL independent (as observed for LacY), frequent and CL dependent (as observed for ProP and MscS), or infrequent (as observed for AqpZ, ProW, and MscL).

SUBMITTER: Romantsov T

PROVIDER: S-EPMC2812954 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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Protein localization in Escherichia coli cells: comparison of the cytoplasmic membrane proteins ProP, LacY, ProW, AqpZ, MscS, and MscL.

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