THE MAN NOSE TRANSPORTER OF ESCHERICHIA-COLI K12 - OLIGOMERIC STRUCTURE, AND FUNCTION OF 2 CONSERVED CYSTEINES

The mannose transporter of E. coli is a member of the phosphotransfera se system. It consists of two membrane spanning subunits, IICMan (27.6 4 kDa) and IIDMan (31.02 kDa) and a peripheral subunit IIAB(Man) (35.0 2 kDa). It acts by a mechanism that couples vectorial translocation to phosphorylation of the substrate. The subunit ratio determined from d ensitometric scans of polyacrylamide gels is close to IIAB(2)(Man) IIC 1Man IID2Man. A molecular mass of 100 +/- 20 kDa was calculated from e lectronmicrographs of freeze fractured proteoliposomes containing part icles of the IICMan/IIDMan subcomplex with a mean diameter of 6.3 +/- 1.1 nm. This is most compatible with IICMan:IIDMan subunit composition s of 1:2 (89.7 kDa). Fusion proteins between IICMan and IIDMan were ge nerated, with the subunits connected either by a two-residue linker or a 20 residue Ala Pro rich hinge. The fusion proteins had 5%-15% of co ntrol phosphotransferase activity. The one with the Ala Pro rich linke r could be cleaved with trypsin resulting in a 7 fold increase of acti vity while the fusion with the two residue linker was resistant to lim ited trypsinolysis. Taking into account the inside-out orientation of the membrane vesicles the C-terminus of IICMan and the N-terminus of I IDMan are both predicted to be on the cytoplasmic side of the membrane . Two cysteines in IICMan and IIDMan which are conserved in the homolo gous subunits of the fructose transporter of Bacillus subtilis and of sorbose transporter of Klebsiella pneumoniae are not necessary for pho sphotransferase function.