THE GLUCOSE-TRANSPORTER OF ESCHERICHIA-COLI WITH CIRCULARLY PERMUTED DOMAINS IS ACTIVE IN-VIVO AND IN-VITRO

The bacterial phosphotranaferase system (PTS) consists of two energy-c oupling soluble proteins (enzyme I and HPr) and a large number of inne r membrane transporters (enzymes II) that mediate concomitant phosphor ylation and translocation of sugars and hexitols, The transporters con sist of three functional units (IIA, IIB, IIC), which occur either as protein subunits or domains of a multidomain polypeptide, The membrane -spanning HC domain contains the substrate binding site; IIA and IIB a re phosphorylation domains that transfer phosphate from HPr to the tra nsported sugar. The transporter complexes of the PTS are good examples for variation of design by modular assembly of domains and subunits. The domain order is IIC-IIB in the membrane subunit of the Escherichia coli glucose transporter (IICBGlc) and IIB-IIC in Salmonella typhimur ium sucrose transporter (IIBCSer). The phosphorylation domain of IICBG lc was translocated from the carboxyl-terminal to the amino-terminal e nd of the IIC domain, and the activity of the circularly permuted form was optimized by variation of the length and the composition of the i nterdomain linker. IIBapC(Glc) with an alanine-proline-rich interdomai n linker has 70% of the control specific activity after purification a nd reconstitution into proteoliposomes. These results indicate that th e aminoterminal end of IICBGlc must be on the cytoplasmic side of the inner membrane, that membrane insertion of the IIC domain is insensiti ve to the modification of its amino-terminal end, and that a domain sw ap as it could occur by a single DNA translocation event can rapidly l ead to a functional protein, However, IIB could not be substituted for by glucokinase. Fusion proteins between the IIC domain and glucokinas e do not transport and phosphorylate glucose in an ATP dependent mecha nism, although the IIC moiety displays transport activity upon complem entation with soluble subclonal IIB, and the glucokinase moiety retain s ATP-dependent non-vectorial kinase activity. This indicates that IIC and IIB are two cooperative units and not only sequentially acting up on a common substrate, and that translocation of glucose must be confo rmationally coupled to the phosphorylation/dephosphorylation cycle of IIB.