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.

A multidomain protein comprising the four subunits of the glucose phosphotransferase system of Escherichia coli was constructed by fusion of the transmembrane subunit IICBGlc and the three cytoplasmic proteins, IIAGlc, HPr, and enzyme I. The subunits were linked in the above order with Ala-Pro-rich linkers; the fusion protein was overexpressed in E. coli and purified by Ni2+ chelate affinity chromatography. Approximately 3 mg of the fusion protein could be purified from 1 liter of culture. The phosphotransferase activity of the purified fusion protein was 3-4 times higher than that of an equimolar mixture of the isolated subunits. The mannose transporter, which also requires enzyme I and HPr, was not an effective competitor in the overall phosphoryltransfer reaction when the fusion protein was used, whereas it was a competitor when an equimolar mixture of the separate subunits was employed. Transphosphorylation activity of the fusion protein was almost indistinguishable from the wild-type IICBglc. Addition of extra IICBGlc subunit could significantly stimulate the phosphotransferase activity of the fusion protein, addition of extra IIAGlc subunit and enzyme I, in contrast, was slightly inhibitory, and HPr had almost no effect. An optimal detergent-lipid ratio is required for maximum activity of the fusion protein. Our results suggest that Ala-Pro-rich linker sequences may be of general use for the construction of catalytically active fusion proteins with novel properties.

The IIBCG'" transmembrane subunit of the glucose transporter of E coli containing a carboxy-termmal affinity tag consisting of six adjacent histidines was purified by nickel chelate affinity chromatography.The protem was constitutively overexpressed from a high copy number plasmid. 1.5 mg of 95% pure protein was obtained from 5 g (wet weight) cells. 70% ofthe phosphotransferase activity present m cell membranes was recovered. Adsorption to the nickel resin allows dehpidation as well as rapid detergent exchange. The procedure was used to demonstrate exchange of subunits in the IIBCG'C dimer and it helds promise for the Investigation of other protein-protein mteractions Phosphotransferase system: Glucose carrier; Ni' chelate chromatography; Enzyme II: E co/i

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