Antonie van Leeuwenhoek 65: 349-358, 1994. 349 (~) 1994 KluwerAcademicPublishers. Printedin the Netherlands. Dissection of discrete kinetic events in the binding of antibiotics and substrates to the galactose-H + symport protein, GalP, of Escherichia coli Peter J.E Henderson, Giles E.M. Martin, Terence E McDonald, Angela Steel & Adrian R. Walmsley Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK * requests for offprints: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotech- nology, University of Sheffield, Sheffield SIO 2UH, UK Key words: antibiotics, sugar transport, transport, transport protein Abstract GalP is the membrane protein responsible for H +-driven uptake of D-galactose into Escherichia coli. It is suggested to be the bacterial equivalent of the mammalian glucose transporter, GLUT1, since these proteins share sequence homology, recognise and transport similar substrates and are both inhibited by cytochalasin B and forskolin. The successful over-production of GalP to 35-55% of the total inner membrane protein of E. coli has allowed direct physical measurements on isolated membrane preparations. The binding of the antibiotics cytochalasin B and forskolin could be monitored from changes in the inherent fluorescence of Gale enabling derivation of a kinetic mechanism describing the interaction between the ligands and Gale The binding of sugars to GalP produces little or no change in the inherent fluorescence of the transporter. However, the binding of transported sugars to GalP produces a large increase in the fluorescence of 8-anilino-l-naphthalene sulphonate (ANS) excited via tryptophan residues. This has allowed a binding step, in addition to two putative translocation steps, to be measured. From all these studies a basic kinetic mechanism for the transport cycle under non-energised conditions has been derived. The ease of genetical manipulation of the galP gene in E. coli has been exploited to mutate individual amino acid residues that are predicted to play a critical role in transport activity and/or the recognition of substrates and antibiotics. Investigation of these mutant proteins using the fluorescence measurements should elucidate the role of individual residues in the transport cycle as well as refine the current model. Abbreviations: GalP - galactose-H + transporter, AraE - arabinose-H + transporter, GLUT1 - human erythrocyte glucose transporter Introduction In Escherichia coli there are separate transport proteins for galactose-H +, arabinose-H + and xylose-H+ sym- port. They are designated Gale AraE and XylE, respec- tively. GalP and AraE are 64% identical in amino acid sequence to each other (Henderson et al. 1992; Hen- derson 1993) and 33-22% identical with XylE and sugar transporters from diverse organisms, including cyanobacteria, eubacteria, yeasts, algae, plants, pro- tozoans and mammals (Griffith et al. 1992; Baldwin 1993). This level of similarity is sufficient to establish that the three-dimensional structures of these proteins are very similar, with only subtle differences that deter, mine variations in sugar and cation recognition. The E. coli GalP protein is particularly interesting because of the similarity of its substrate specificity to that of the homologous human glucose transport protein, GLUT1 (Barnett et al. 1973; Walmsley et al. 1994b); this sim- ilarity is reinforced by the susceptibility of both GalP and GLUT1 to inhibition by the antibiotics cytocha- lasin B (Jung & Rampal 1977; Cairns et al. 1991; Walmsley et al. 1994b) and forskolin (Laurenza et al. 1989; Sergeant & Kim 1985; Joost et al. 1988; Martin et al. 1994a). The GalP protein is, therefore, a struc-