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A novel outer membrane lipoprotein, LolB (HemM), involved in the LolA (p20)-dependent localization of lipoproteins to the outer membrane of Escherichia coli.

ABSTRACT: The Escherichia coli major outer membrane lipoprotein (Lpp) is released from the inner membrane into the periplasm as a complex with a carrier protein, LolA (p20), and is then specifically incorporated into the outer membrane. An outer membrane protein playing a critical role in Lpp incorporation was identified, and partial amino acid sequences of the protein, named LolB, were identical to those of HemM, which has been suggested to play a role in 5-aminolevulinic acid synthesis in the cytosol. In contrast to this suggested role, the deduced amino acid sequence of HemM implied that the gene encodes a novel outer membrane lipoprotein. Indeed, an antibody raised against highly purified LolB revealed its outer membrane localization, and inhibited in vitro Lpp incorporation into the outer membrane. Furthermore, LolB was found to be synthesized as a precursor with a signal sequence and then processed to a lipid-modified mature form. An E.coli strain possessing chromosomal hemM under the control of the lac promoter-operator required IPTG for growth, indicating that hemM (lolB) is an essential gene. Outer membrane prepared from LolB-depleted cells did not incorporate Lpp. When the Lpp-LolA complex was incubated with a water-soluble LolB derivative, Lpp was transferred from LolA to LolB. Based on these results, the outer membrane localization pathway for E.coli lipoprotein is discussed with respect to the functions of LolA and LolB.

SUBMITTER: Matsuyama S

PROVIDER: S-EPMC1170298 | biostudies-other | 1997 Dec

REPOSITORIES: biostudies-other

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A novel outer membrane lipoprotein, LolB (HemM), involved in the LolA (p20)-dependent localization of lipoproteins to the outer membrane of Escherichia coli.

Matsuyama S i Si Yokota N N Tokuda H H

The EMBO journal 19971201 23

The Escherichia coli major outer membrane lipoprotein (Lpp) is released from the inner membrane into the periplasm as a complex with a carrier protein, LolA (p20), and is then specifically incorporated into the outer membrane. An outer membrane protein playing a critical role in Lpp incorporation was identified, and partial amino acid sequences of the protein, named LolB, were identical to those of HemM, which has been suggested to play a role in 5-aminolevulinic acid synthesis in the cytosol. I ...[more]

PMID: 9384574

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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

A novel outer membrane lipoprotein, LolB (HemM), involved in the LolA (p20)-dependent localization of lipoproteins to the outer membrane of Escherichia coli.

Publications

A novel outer membrane lipoprotein, LolB (HemM), involved in the LolA (p20)-dependent localization of lipoproteins to the outer membrane of Escherichia coli.

Similar Datasets

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