Isolation and identification of new inner membrane-associated proteins that localize to cell poles in Escherichia coli

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. TnaAGFP and GroESGFP 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.