SummaryA new family of homologous membrane proteins that transport galactosides-pentoses-hexuronides (GPH) is described. By analysing the aligned amino acid sequences of the GPH family, and by exploiting their different specificities for cations and sugars, we have designed mutations that yield novel insights into the nature of ligand binding sites in membrane proteins. Mutants have been isolated/constructed in the melibiose transport proteins of Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium, and the lactose transport protein of Streptococcus thermophilus which facilitate uncoupled transport or have an altered cation and/or substrate specificity. Most of the mutations map in the amino-terminal region, in or near amphipathic a -helices II and IV, or in interhelix-loop 10-11 of the transport proteins. On the basis of the kinetic properties of these mutants, and the primary and secondary structure analyses presented here, we speculate on the cation binding pocket of this family of transporters. The regulation of the transporters through interaction with, or phosphorylation by, components of the phosphoenolpyruvate:sugar phosphotransferase system is also discussed.

As much as 20-30 mg of functional recombinant melibiose permease (Mel-6His permease) of Escherichia coli, carrying a carboxy-terminal affinity tag for metallic ions (six successive histidines), can be routinely purified from 10 g of cells (dry weight) by combining nickel chelate affinity chromatography and ion exchange chromatography. Mel-6His permease was constructed by modifying the permease gene (melB) in vitro and then overproduced in cells transformed with multicopy plasmids. The tagged permease was efficiently solubilized in the presence of 3-(laurylamido)-N,N'-dimethylaminopropylamine oxide (LAPAO) and high sodium salt concentration and then selectively adsorbed on a nickel nitrilotriacetic acid (Ni-NTA) affinity resin. After the replacement of LAPAO by n-dodecyl beta-D-maltoside to maintain the activity of the soluble permease in low ionic strength media, the permease-enriched fraction (> 90%) was eluted with 0.1 M imidazole and finally purified to homogeneity (> 99%) using ion exchange chromatography. Determination of the permease N-terminal sequence shows that an initiating methionine is missing and that a Ser-Ile-Ser stretch precedes the postulated primary amino acid sequence. Purified permeases, reconstituted in liposomes, display H(+)-, Na(+)-, or Li(+)-dependent sugar binding and active transport activities similar to those of the native permease in its natural environment, proving that all three modes of symport activity are mediated by one and the same polypeptide.

Tryptophan fluorescence spectroscopy has been used to investigate the effects of sugars and coupling cations (H+, Na+, or Li+) on the conformational properties of purified melibiose permease after reconstitution in liposomes. Melibiose permease emission fluorescence is selectively enhanced by sugars, which serve as substrates for the symport reaction, alpha-galactosides producing larger variations (13-17%) than beta-galactosides (7%). Moreover, the sugar-dependent fluorescence increase is specifically potentiated by NaCl and LiCl (5-7 times), which are well-established activators of sugar binding and transport by the permease. The potentiation effect is greater in the presence of LiCl than NaCl. On their own, sodium and lithium ions produce quenching of the fluorescence signal (2%). Evidence suggesting that sugars and cations compete for their respective binding sites is also given. Both the sugar-induced fluorescence variation and the NaCl(or LiCl)-dependent potentiation effect exhibit saturation kinetics. In each ionic condition, the half-maximal fluorescence change is found at a sugar concentration corresponding to the sugar-binding constant. Also, half-maximal potentiation of the fluorescence change by sodium or lithium occurs at a concentration comparable to the activation constant of sugar binding by each ion. The sugar- and ion-dependent fluorescence variations still take place after selective inactivation of the permease substrate translocation capacity by N-ethylmaleimide. Taken together, the data suggest that the changes in permease fluorescence reflect conformational changes occurring upon the formation of ternary sugar/cation/permease complexes.

In order to study the secondary structure of the melibiose permease of Escherichia coli, 57 melB-phoA gene fusions were constructed and assayed for alkaline phosphatase activity. In general agreement with a previously suggested secondary structure model of melibiose permease [Botfield, M. C., Naguchi, K., Tsuchiya, T., & Wilson, T.H. (1992) J. Biol. Chem. 267, 1818], clusters of fusions exhibiting low and high phosphatase activity fusions alternate along the primary sequence. Fusions with high activity generally cluster at residues predicted to be in the periplasmic half of transmembrane domains or in periplasmic loops, while fusions with low activity cluster at residues predicted to be in the cytoplasmic half of transmembrane domains or in cytoplasmic loops. Taken together, the findings strongly support the contention that melibiose permease contains 12 transmembrane domains that traverse the membrane in zigzag fashion connected by hydrophilic loops that are exposed alternatively on the periplasmic or cytoplasmic surfaces of the membrane with the N and C termini on the cytoplasmic face of the membrane. Moreover, on the basis of the finding that the cytoplasmic half of an out-going segment is sufficient for alkaline phosphatase export to the periplasm while the periplasmic half of an in-going segment prevents it [Calamia, T., & Manoil, C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4937], the activity profile of the melibiose permease-alkaline phosphatase fusions is consistent with the predicted topology of seven of 12 transmembrane segments. However, five transmembrane domains require adjustment, and as a consequence, the size of the central cytoplasmic loop is reduced and a significant number of charged residues are shifted from a hydrophilic to a hydrophobic domain in this region of the transporter.