Membrane localization itself but not binding to IICB Glc is directly responsible for the inactivation of the global repressor Mlc in Escherichia coli

Summary Mlc is a global transcriptional repressor involved in the regulation of genes linked to glucose metabolism. The activity of Mlc is modulated through the interaction with a major glucose transporter, IICB Glc , in response to external glucose. To understand how IICB Glc –Mlc interaction controls the repressor activity of Mlc, we attempted to isolate Mlc mutants that retain the ability to repress target genes even in the presence of glucose. The Mlc mutants were tested for their ability to interact with IICB Glc . Mutants in which a single amino acid substitution occurs in the N‐terminal portion were no longer able to bind to IICB Glc , suggesting that the N‐terminal region of Mlc is primarily responsible for the interaction with IICB Glc . To examine whether the Mlc–IICB Glc interaction and/or the membrane localization of Mlc per se are essential for the inactivation of Mlc, the properties of several hybrid proteins in which either IIB Glc or Mlc is fused to membrane proteins were analysed. The cytoplasmic IIB Glc domain failed to inhibit the Mlc action although it retains the ability to bind Mlc in cells. However, it gained the ability to inhibit the Mlc activity when it was fused to a membrane protein LacY. In addition, we showed that Mlc is inactivated when fused to membrane proteins but not when fused to cytoplasmic proteins. We conclude that the IICB Glc –Mlc interaction is dispensable for the inactivation of Mlc, and that membrane localization is directly responsible for the inactivation of Mlc.