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 similar in substrate specificity and susceptibility to cytochalasin B and forskolin, to the human GLUT1 sugar-transport protein; furthermore, they are about 30% identical in amino acid sequence. Transport activities of both GalP and GLUT1 are inhibited by the thiol-group-specific reagent, N-ethylmaleimide. 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 activity, whereas alteration of all three simultaneously profoundly diminishes V(max) for transport. The high level of expression of the GalP protein facilitates measurements of the reactivity of each mutant with N-ethylmaleimide or eosin 5-maleimide, which conclusively demonstrate that Cys(374) is the site of covalent modification by the reagents. By comparing the reactivity of Cys(374) in right-side-out and inside-out vesicles it appears that Cys(374) is located on the cytoplasmic face of the GalP protein. Although impaired in transport activity, the 'Cys-free' mutant, with all three cysteine residues mutated into serine, binds cytochalasin B and forskolin with wild-type affinities. All these results are interpreted in terms of a 12-helix model of the folding of the protein, in which the relative orientations of helix 10, containing the reactive Cys(374) residue, and helix 11, containing the unreactive Cys(389) residue, can now be defined.
McDonald
T P
TP
School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Henderson
P J
PJ
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D002135:Calcium-Binding Proteins; D005576:Colforsin / Q000378:metabolism; D003545:Cysteine / Q000235:genetics*; D003571:Cytochalasin B / Q000378:metabolism; D017931:DNA Primers; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives / Q000378:metabolism; D005033:Ethylmaleimide / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins; D011485:Protein Binding
D000900:Anti-Bacterial Agents; D002135:Calcium-Binding Proteins; D017931:DNA Primers; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C023550:galactose-binding protein; D005576:Colforsin; D003571:Cytochalasin B; C033531:eosin maleimide; D003545:Cysteine; D005033:Ethylmaleimide; D004801:Eosine Yellowish-(YS); D005690:Galactose