MEMBRANE TOPOLOGY OF THE MANNOSE TRANSPORTER OF ESCHERICHIA-COLI K12

The mannose transporter of the bacterial phosphotransferase system med iates carbohydrate transport across the cytoplasmic membrane concomita nt with carbohydrate phosphorylation. It also functions as a receptor for bacterial chemotaxis [Adler, J. & Epstein, W. (1974) Proc. Natl Ac ad. Sci. USA 71, 2895-2899] and is required for infection of the cell by bacteriophage lambda where it most likely functions as a pore for p enetration of phage DNA [Elliott, J. & Arber, W. (1978) Mol. & Gen. Ge net. 161, 1-8]. The transporter consists of two transmembrane subunits (27-kDa IICMan and 31-kDa IIDMan) and a hydrophilic subunit (35-kDa I IAB(Man)). Protein fusions of IICMan and IIDMan with beta-galactosidas e (LacZ) and with alkaline phosphatase (PhoA) were analyzed to determi ne the membrane topology of the two proteins. Protein fusions were obt ained by progressively deleting the manY and manZ genes from their 3' ends and ligating them to lacZ and 'phoA that lack promotor and leader sequences. Based on the analysis of 30 IICMan-PhoA, 10 IICMan-LacZ, 1 2 IIDMan-PhoA, and 30 IIDMan-LacZ fusions, it is predicted that IICMan has six membrane-spanning segments with the N- and C-termini on the c ytoplasmic face of the membrane. IIDMan is anchored in the membrane by a single membrane-spanning segment at the end of the C-terminus, whil e most of the protein (250 residues) protrudes into the cytoplasm.