FUNCTIONAL RECONSTITUTION OF THE PURIFIED MANNOSE PHOSPHOTRANSFERASE SYSTEM OF ESCHERICHIA-COLI INTO PHOSPHOLIPID-VESICLES

The mannose transporter complex acts by a mechanism which couples tran slocation with phosphorylation of the substrate. It consists of a hydr ophilic subunit (IIAB(Man)) and two transmembrane subunits (IICMan, II DMan). The purified complex was reconstituted into phospholipid vesicl es by octyl glucoside dilution. Glucose export was measured with prote oliposomes which were loaded with radiolabeled glucose and to which pu rified ILAB(Man), cytoplasmic phosphorylcarrier proteins, and P-enolpy ruvate were added from the outside. Vectorial transport was accompanie d by stoichiometric phosphorylation of the transported sugar. Glucose added to the outside of the proteoliposomes was also phosphorylated ra pidly but did not compete with vectorial export and phosphorylation of internal glucose. Glucose uptake was measured with proteoliposomes wh ich were loaded with the cytoplasmic phosphoryl carrier proteins and P -enolpyruvate and to which glucose was added from the outside. Vectori al import and phosphorylation occurred with a higher specificity (K-m 30 +/- 6 mu M, k(cat) 401 +/- 32 pmol of Glc/mu g of IICDMan/min) than nonvectorial phosphorylation (K-m 201 +/- 43 mu M, k(cat) 975 +/- 88 pmol of Glc/mu g of IICDMan/min). A new plasmid pTSHIC9 for the contro lled overexpression of the cytoplasmic phosphoryl carrier proteins, en zyme I, HPr, and IIA(Glc), and a simplified procedure for the purifica tion of these proteins are also described.