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 (IIBCGlc) and a hydrophilic subunit (IIAGlc). Both subunits are transiently phosphorylated. The IIBCGlc 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 IIBCGlc and beta-galactosidase (LacZ) as well as alkaline phosphatase (PhoA) were analyzed to determine the membrane topology of the IIBCGlc 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 IICGlc (residues 19-336) is suggested to contains eight membrane-spanning segments, with the amino terminus and the COOH-terminal hydrophilic domain (IIBGlc) located on the cytoplasmic face of the membrane. A sequence comparison of IIBCGlc with three related proteins indicates that the periplasmic loops differ in size and sequence while the cytoplasmic loops are better conserved.
Buhr
A
A
Institute for Biochemistry, University of Bern, Switzerland.
Erni
B
B
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000235:genetics; D011401:Promoter Regions, Genetic; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics
D046911:Macromolecular Substances; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase