MEMBRANE TOPOLOGY OF THE GLUCOSE TRANSPORTER OF ESCHERICHIA-COLI

The glucose transporter of the bacterial phosphotransferase system cou ples translocation with phosphorylation of the substrate in a 1:1 stoi chiometry. It consists of a transmembrane subunit (IIBC(Glc)) and a hy drophilic subunit (IIA(Glc)). Both subunits are transiently phosphoryl ated. The IIBC(Glc) subunit is 477 residues long and consists of two d omains. The amino-terminal hydrophobic domain is involved in glucose b inding and translocation, the carboxyl-terminal domain contains the ph osphorylation site (Cys421). Protein fusions between IIBC(Glc) and bet a-galactosidase (LacZ) as well as alkaline phosphatase (PhoA) were ana lyzed to determine the membrane topology of the IIBC(Glc) subunit. The protein fusions were generated by progressively deleting ptsG from it s 3' end and ligating the truncated gene to lacZ and 'phoA lacking pro moter and leader sequences. LacZ fusions of high activity (32 out of 5 4) occur at the amino and carboxyl termini and three internal clusters , and 41 active PhoA fusions occur in four internal clusters. Accordin gly 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 cyt oplasmic 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.