Cysteine residues in the D-galactose-H+ symport protein of Escherichia coli: effects of mutagenesis on transport, reaction with N-ethylmaleimide and antibiotic binding

The galactose-H+ membrane-transport protein, GalP, of Escherichia coli is s imilar in substrate specificity and susceptibility to cytochalasin B and fo rskolin, to the human GLUT1 sugar-transport protein; furthermore, they are about 30 % identical in amino acid sequence. Transport activities of both G alP and GLUT1 are inhibited by the thiol-group-specific reagent, N-ethylmal eimide. GalP contains only three cysteine residues at positions 19, 374 and 389, each of which we have mutated, singly and in combination, to serine. Each single change of Cys --> Ser has only a minor effect on transport acti vity, whereas alteration of all three simultaneously profoundly diminishes V-max for transport. The high level of expression of the GalP protein facil itates measurements of the reactivity of each mutant with N-ethylmaleimide or eosin 5-maleimide. which conclusively demonstrate that Cys(374) is the s ite of covalent modification by the reagents. By comparing the reactivity o f Cys(374) in right-side-out and inside-out vesicles it appears that Cys(37 4) is, located on the cytoplasmic face of the GalP protein. Although impair ed in transport activity. the 'Cys-free' mutant, with all three cysteine re sidues mutated into serine, binds cytochalasin B and forskolin with wild-ty pe affinities. All these results are interpreted in terms of a 12-helix mod el of the folding of the protein, in which the relative orientations of hel ix 10, containing the reactive Cys(374) residue, and helix 11, containing t he unreactive Cys(389) residue, can now be defined.