The mannose transporter of Escherichia coli K12: oligomeric structure, and function of two conserved cysteines.
The mannose transporter of E. coli is a member of the phosphotransferase system. It consists of two membrane spanning subunits, IICMan (27.64 kDa) and IIDMan (31.02 kDa) and a peripheral subunit IIABMan (35.02 kDa). It acts by a mechanism that couples vectorial translocation to phosphorylation of the substrate. The subunit ratio determined from densitometric scans of polyacrylamide gels is close to IIABMan2 IICMan1 IIDMan2. A molecular mass of 100 +/- 20 kDa was calculated from electronmicrographs of freeze fractured proteoliposomes containing particles of the IICMan/IIDMan subcomplex with a mean diameter of 6.3 +/- 1.1 nm. This is most compatible with IICMan:IIDMan subunit compositions of 1:2 (89.7 kDa). Fusion proteins between IICMan and IIDMan were generated, 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 control phosphotransferase activity. The one with the Ala Pro rich linker could be cleaved with trypsin resulting in a 7 fold increase of activity while the fusion with the two residue linker was resistant to limited trypsinolysis. Taking into account the inside-out orientation of the membrane vesicles the C-terminus of IICMan and the N-terminus of IIDMan are both predicted to be on the cytoplasmic side of the membrane. Two cysteines in IICMan and IIDMan which are conserved in the homologous subunits of the fructose transporter of Bacillus subtilis and of sorbose transporter of Klebsiella pneumoniae are not necessary for phosphotransferase function.
Rhiel
E
E
Fachbereich Biologie der Philipps-Universität Marburg, Germany.
Flükiger
K
K
Wehrli
C
C
Erni
B
B
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D001483:Base Sequence; D001693:Biological Transport, Active; D003545:Cysteine / Q000737:chemistry / Q000502:physiology*; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D005614:Freeze Fracturing; D005632:Fructose / Q000378:metabolism; D007525:Isoelectric Focusing; D008358:Mannose / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism
D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D011510:Proteolipids; C020485:proteoliposomes; D005632:Fructose; D010770:Phosphotransferases; D003545:Cysteine; D008358:Mannose