Functional reconstitution of the purified mannose phosphotransferase system of Escherichia coli into phospholipid vesicles.
The mannose transporter complex acts by a mechanism which couples translocation with phosphorylation of the substrate. It consists of a hydrophilic subunit (IIABMan) and two transmembrane subunits (IICMan, IIDMan). The purified complex was reconstituted into phospholipid vesicles by octyl glucoside dilution. Glucose export was measured with proteoliposomes which were loaded with radiolabeled glucose and to which purified IIABMan, cytoplasmic phosphorylcarrier proteins, and P-enolpyruvate were added from the outside. Vectorial transport was accompanied by stoichiometric phosphorylation of the transported sugar. Glucose added to the outside of the proteoliposomes was also phosphorylated rapidly but did not compete with vectorial export and phosphorylation of internal glucose. Glucose uptake was measured with proteoliposomes which were loaded with the cytoplasmic phosphoryl carrier proteins and P-enolpyruvate and to which glucose was added from the outside. Vectorial import and phosphorylation occurred with a higher specificity (Km 30 +/- 6 microM, kcat 401 +/- 32 pmol of Glc/micrograms of IICDMan/min) than nonvectorial phosphorylation (Km 201 +/- 43 microM, kcat 975 +/- 88 pmol of Glc/micrograms of IICDMan/min). A new plasmid pTSHIC9 for the controlled overexpression of the cytoplasmic phosphoryl carrier proteins, enzyme I, HPr, and IIAGlc, and a simplified procedure for the purification of these proteins are also described.
Mao
Q
Q
Institute for Biochemistry, University of Bern, Switzerland.
Schunk
T
T
Flükiger
K
K
Erni
B
B
D004926:Escherichia coli / Q000201:enzymology*; D015870:Gene Expression; D007700:Kinetics; D008081:Liposomes; D046911:Macromolecular Substances; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011510:Proteolipids / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion
D008081:Liposomes; D046911:Macromolecular Substances; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C037992:phosphoenolpyruvate-mannose phosphotransferase