Cytoplasmic loop connecting helices IV and V of the melibiose permease from Escherichia coli is involved in the process of Na+-coupled sugar translocation.
Previous photolabeling and limited proteolysis studies suggested that one of the four basic residues (Arg-141) of the N-terminal cytoplasmic loop connecting helices IV and V (loop 4-5) of the melibiose permease (MelB) from Escherichia coli has a potential role in its symport function (Ambroise, Y., Leblanc, G., and Rousseau, B. (2000) Biochemistry 39, 1338-1345). A mutagenesis study of Arg-141 and of the other three basic residues of loop 4-5 was undertaken to further examine this hypothesis. Cys replacement analysis indicated that Arg-141 and Arg-149, but not Lys-138 and Arg-139, are essential for MelB transport activity. Replacement of Arg-141 by neutral residues (Cys or Gln) inactivated transport and energy-independent carrier-mediated flows of substrates (counterflow, efflux), whereas it had a limited effect on co-substrate binding. R141C sugar transport was partially rescued on reintroducing a positive charge with a charged and permeant thiol reagent. Whereas R149C was completely inactive, R149K and R149Q remained functional. Strikingly, introduction of an additional mutation in the C-terminal helix X (Gly for Val-343) of R149C restored sugar transport. Impermeant thiol reagents inhibited R149C/V343G transport activity in right-side-out membrane vesicles and prevented sugar binding in a sugar-protected manner. All these data suggest that MelB loop 4-5 is close to the sugar binding site and that the charged residue Arg-141 is involved in the reaction of co-substrate translocation or substrate release in the inner compartment.
Abdel-Dayem
Manal
M
Laboratoire de Physiologie des Membranes Cellulaires, Université de Nice Sophia-Antipolis and CNRS UMR 6078, Commissariat à l'Energie Atomique (LRC-CEA 16V), Villefranche sur mer, 06230 France.
Basquin
Cécile
C
Pourcher
Thierry
T
Cordat
Emmanuelle
E
Leblanc
Gérard
G
D000595:Amino Acid Sequence; D001665:Binding Sites; D001692:Biological Transport; D050260:Carbohydrate Metabolism; D003593:Cytoplasm / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D012964:Sodium / Q000378:metabolism*; D027981:Symporters / Q000737:chemistry / Q000378:metabolism*
D027981:Symporters; C021548:melibiose permease; D012964:Sodium