Interactions of the chemotaxis signal protein CheY with bacterial flagellar motors visualized by evanescent wave microscopy.
The chemotaxis signal protein CheY of enteric bacteria shuttles between transmembrane methyl-accepting chemotaxis protein (MCP) receptor complexes and flagellar basal bodies [1]. The basal body C-rings, composed of the FliM, FliG and FliN proteins, form the rotor of the flagellar motor [2]. Phosphorylated CheY binds to isolated FliM [3] and may also interact with FliG [4], but its binding to basal bodies has not been measured. Using the chemorepellent acetate to phosphorylate and acetylate CheY [5], we have measured the covalent-modification-dependent binding of a green fluorescent protein-CheY fusion (GFP-CheY) to motor assemblies in bacteria lacking MCP complexes by evanescent wave microscopy [6]. At acetate concentrations that cause solely clockwise rotation, GFP-CheY molecules bound to native basal bodies or to overproduced rotor complexes with a stoichiometry comparable to the number of C-ring subunits. GFP-CheY did not bind to rotors lacking FIiM/FliN, showing that these subunits are essential for the association. This assay provides a new means of monitoring protein-protein interactions in signal transduction pathways in living cells.
Khan
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Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA. skhan@aecom.yu.edu
Pierce
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Vale
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D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002633:Chemotaxis; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D029968:Escherichia coli Proteins; D005407:Flagella / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D011993:Recombinant Fusion Proteins; C000607606:cheY protein, E coli; D049452:Green Fluorescent Proteins