Interaction of unphosphorylated PtsN with the K/H antiporter YcgO inhibits its activity in Escherichia coli.

Genetic studies in Escherichia coli have implicated the unphosphorylated version of PtsN (unphospho-PtsN), the terminal phospho-acceptor of the PtsP-PtsO-PtsN phosphorelay, as a negative regulator of potassium (K) efflux mediated by YcgO. YcgO is a protein belonging to the CPA1 family of monovalent cation/proton antiporters. Here we show that in vivo, YcgO comprises an approximately 383 amino acid N-terminal transmembrane domain and a 195 amino acid C-terminal cytoplasmic region (CTR). Copurification studies show that unphospho-PtsN specifically interacts with YcgO, and phosphorylation of PtsN leads to marked attenuation of the interaction. Genetic and biochemical analyses of a class of mutations in YcgO that lead to constitutive activation of YcgO identify the CTR as the site of interaction between unphospho-PtsN and YcgO and indicate that the putative CorC domain in the CTR may serve as the site of interaction. Our studies are supportive of a model which postulates that the unphospho-PtsN:CorC interaction may inhibit the activation of YcgO by a putative RCK domain in the CTR, leading to the inhibition of the K/H antiport activity of YcgO.