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Localization of a putative second membrane association site in penicillin-binding protein 1B of Escherichia coli.

ABSTRACT: We have shown previously that the periplasmic domain of penicillin-binding protein 1B (PBP 1Bper; residues 90-844) from Escherichia coli is insoluble in the absence of detergents, and can be reconstituted into liposomes [Nicholas, Lamson and Schultz (1993) J. Biol. Chem. 268, 5632-5641]. These data suggested that native PBP 1B contains a membrane association site in addition to its N-terminal transmembrane anchor. We have studied the membrane topology of PBP 1B in greater detail by assessing detergent binding and solubility in the absence of detergents for PBP 1Bper and a set of proteolytic fragments of PBP 1B. PBP 1Bper was shown by three independent methods to bind to detergent micelles, which strongly suggests that the periplasmic domain interacts with the hydrophobic milieu of membrane bilayers. Digestion with high weight ratios of thrombin of purified PBP 1B containing an engineered thrombin cleavage site on the periplasmic side of the transmembrane anchor generated four fragments in addition to PBP 1Bper that varied in size from 71 to 48 kDa. In contrast to PBP 1Bper, all fragments of 67 kDa and smaller were eluted from a gel-filtration column in the absence of detergents and did not bind to detergent micelles. The N-terminal sequences of the four fragments were determined, allowing the cleavage sites to be located in the primary sequence of PBP 1B. These data localize the membrane association site of PBP 1B to a region comprising the first 163 amino acids of the periplasmic domain, which falls within the putative transglycosylase domain. Lipid modification does not appear to be the mechanism by which PBP 1Bper associates with membranes.

SUBMITTER: Wang CC

PROVIDER: S-EPMC1217315 | biostudies-other | 1996 May

REPOSITORIES: biostudies-other

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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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Localization of a putative second membrane association site in penicillin-binding protein 1B of Escherichia coli.

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