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Isolation and identification of new inner membrane-associated proteins that localize to cell poles in Escherichia coli.
Mol Microbiol. 2012 Apr; 84(2):276-95
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23 Mar 2012
Technical Advance
DOI: 10.3410/f.14208996.15726119
This paper describes an innovative method for identifying proteins enriched at bacterial cell poles. Cell poles play an important role in bacterial cell physiology, serving as localization sites for factors including secretion machinery, flagellar apparatus, pili and a host of virulence factors. How how...Continue reading
This paper describes an innovative method for identifying proteins enriched at bacterial cell poles. Cell poles play an important role in bacterial cell physiology, serving as localization sites for factors including secretion machinery, flagellar apparatus, pili and a host of virulence factors. However, how these proteins target the pole is not well understood. Taking advantage of FLAG-tagged variants of the polarly localized chemotaxis protein Tar and an anti-FLAG antibody attached to agarose beads, Li and Young enriched for membrane vesicles containing polar components. Mass spectrometry of these polar vesicles identified 31 proteins, many of which had not previously been known to concentrate at cell poles.
This method is broadly applicable and represents a significant improvement on other approaches for identifying proteins enriched at cell poles. Li and Young's approach will thus increase the list of proteins with polar localization patterns, and, through comparative analysis, could potentially lead to the identification of determinants required for polar localization in Escherichia coli and other bacteria.
This method is broadly applicable and represents a significant improvement on other approaches for identifying proteins enriched at cell poles. Li and Young's approach will thus increase the list of proteins with polar localization patterns, and, through comparative analysis, could potentially lead to the identification of determinants required for polar localization in Escherichia coli and other bacteria.
Disclosures
None declared
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Levin P: F1000Prime Recommendation of [Li G and Young KD, Mol Microbiol 2012, 84(2):276-95]. In F1000Prime, 23 Mar 2012; DOI: 10.3410/f.14208996.15726119. F1000Prime.com/14208996#eval15726119
F1000Prime Recommendations, Dissents and Comments for [Li G and Young KD, Mol Microbiol 2012, 84(2):276-95]. In F1000Prime, 19 May 2013; F1000Prime.com/14208996
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Several bacterial structures, processes and proteins are localized primarily to the poles of rod-shaped cells. To better understand this cellular organization, we devised a new method for identifying proteins that localize to the poles of Escherichia coli. Pole-derived membrane fragments were isolated by affinity capture of vesicles containing the chemotaxis protein, Tar; and for comparison, vesicles representing all parts of the cytoplasmic membrane were captured by expressing a Tar variant that was no longer pole-specific. A combination of one-dimensional SDS-PAGE and semi-quantitative mass spectrometry identified 31 proteins that were highly enriched in polar vesicles. Five were chemotaxis proteins known to be pole-specific and another, Aer, was an aerotaxis protein that had not yet been localized to the pole. The behaviour of these internal controls validated the overall approach. GFP-fused derivatives of four candidates (Aer, YqjD, TnaA and GroES) formed polar foci that were distinct from inclusion bodies. TnaA-GFP and GroES-GFP were functional, formed a single focus per cell, and competed for polar localization with the wild-type versions of these proteins. Polar localization of TnaA, GroES and YqjD was disrupted in cells lacking the MinCDE proteins, suggesting that this system may help localize proteins not involved in cell division.
© 2012 Blackwell Publishing Ltd.
DOI: 10.1111/j.1365-2958.2012.08021.x
PMID: 22380631
