Abstract
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.
Highlights
From the slnstitute for Biochemistry, University of Bern, CH-3012 Bern Switzerland and the §Department of Biology, Philipps-University Marburg, D-3550 Marburg, Germany
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
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
Summary
From the slnstitute for Biochemistry, University of Bern, CH-3012 Bern Switzerland and the §Department of Biology, Philipps-University Marburg, D-3550 Marburg, Germany. The mannose transporter complex acts by a mechanism which couples translocation with phosphorylation of the substrate. It consists of a hydrophilic subunit (lIABMan) and two transmembrane subunits (HcMan, HDMan). 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. 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. Uptake of hexoses and hexitols in bacteria is mediated by membrane protein complexes, the so-called enzymes II of the bacterial phosphotransferase system (PTS).l They couple vectorial translocation with phosphorylation of the transported solute.
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