The Glucose Transporter of Escherichia coli

The glucose transporter of the bacterial phosphotransferase system couples vectorial translocation to phosphorylation of the transported sugar. It consists of a transmembrane subunit (IICBGIc) and a hydrophilic subunit (IIAG1"). The IICBGIC subunit consists of two domains. The NH,-terminal IIC domain (residues 1-386) spans the membrane eight times and contains the substrate binding site. The COOH-terminal hydrophilic IIB domain (residues 391476) is accessible from the cytoplasmic side of the membrane. It contains the phosphorylation site (CYS~~') and together with the IIC domain catalyzes the transfer of phosphoryl groups from the IIAGlC subunit to the transported solute. Starting from a plasmid vector containing ptsG under an inducible promoter, the IIB and the IIC domains have been subcloned separately, overexpressed in Escherichia coli, and purified by Ni2+ chelate affinity chromatography. Approximately 40 mg of IIBG1"-6H and 4 mg of IICGIC-6H could be purified from 1 liter of culture. Cells expressing IIBG1"-6H and IICG1"-6H separately have a three times longer generation time on glucose minimal medium than cells expressing wild-type IICBG1". The rate of IIBG'"-6H phosphorylation determined in a nitrocellulose filter binding assay is indistinguishable from wild-type IICBG1". The in vitro specific activity of IICGlC-6H in the presence of excess IIBG1"-6H is 2% of the control. IIBG1"-6H also complements the activity of a IICBG1" mutant with an inactive IIB domain (C421S) indicating that IIC and IIB are flexibly linked such that a free IIB domain can displace an inactive IIB domain from its contact site on the IIC domain. Based on this work, the secondary structure of the IIBGL domain has been determined by isotopeedited NMR spectroscopy (Golic Grdadolnik, s., Eberstadt, M., Gemmecker, G., Kessler, H., Buhr, A., and Erni, B. (1994) Euc J. Biochern. 219,945-952).