MEMBRANE TOPOLOGY OF THE GLUCOSE TRANSPORTER OF ESCHERICHIA-COLI

The glucose transporter of the bacterial phosphotransferase system couples translocation with phosphorylation of the substrate in a 1:1 stoichiometry. It consists of a transmembrane subunit (IIBC(Glc)) and a hydrophilic subunit (IIA(Glc)). Both subunits are transiently phosphorylated. The IIBC(Glc) subunit is 477 residues long and consists of two domains. The amino-terminal hydrophobic domain is involved in glucose binding and translocation, the carboxyl-terminal domain contains the phosphorylation site (Cys421). Protein fusions between IIBC(Glc) and beta-galactosidase (LacZ) as well as alkaline phosphatase (PhoA) were analyzed to determine the membrane topology of the IIBC(Glc) subunit. The protein fusions were generated by progressively deleting ptsG from its 3' end and ligating the truncated gene to lacZ and 'phoA lacking promoter and leader sequences. LacZ fusions of high activity (32 out of 54) occur at the amino and carboxyl termini and three internal clusters, and 41 active PhoA fusions occur in four internal clusters. Accordingly the hydrophobic domain of IIC(Glc) (residues 19-336) is suggested to contains eight membrane-spanning segments, with the amino terminus and the COOH-terminal hydrophilic domain (IIB(Glc)) located on the cytoplasmic face of the membrane. A sequence comparison of IIBC(Glc) with three related proteins indicates that the periplasmic loops differ in size and sequence while the cytoplasmic loops are better conserved.