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 tra nsmembrane 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]. Phosphor ylated 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 chemorepe llent acetate to phosphorylate and acetylate CheY [5], we have measured the covalent-modification-dependent binding of a green fluorescent protein-Che Y fusion (GFP-CheY) to motor assemblies in bacteria lacking MCP complexes b y evanescent wave microscopy [6], At acetate concentrations that cause sole ly clockwise rotation, GFP-CheY molecules bound to native basal bodies or t o overproduced rotor complexes with a stoichiometry comparable to the numbe r of C-ring subunits. GFP-CheY did not bind to rotors lacking FliM/FliN, sh owing that these subunits are essential for the association. This assay pro vides a new means of monitoring protein-protein interactions in signal tran sduction pathways in living cells. (C) 2000 Elsevier Science Ltd. All right s reserved.