Mutagenesis of acidic residues in putative membrane-spanning segments of the melibiose permease of Escherichia coli. II. Effect on cationic selectivity and coupling properties.
Individual substitution of Cys or Asn for Asp-31, Asp-51, Asp-55, or Asp-120, distributed in different membrane spanning segments of the NH2-terminal domain of melibiose (mel) permease partially or completely inactivates Na(+)-linked sugar transport and stimulation of sugar binding on mel permease by Na+ ions (Pourcher, T., Zani, M.-L., and Leblanc, G. (1993) J. Biol. Chem. 268, 3209-3215). To investigate further the effect of these substitutions on the cationic selectivity and coupling properties of mel permease, H(+)-melibiose coupled transport, coupling between H+ and melibiose movements, sugar counterflow, and zero-trans sugar efflux by the mutant permeases were analyzed. The results provide additional evidence indicating that manipulation of some of these Asp in the membrane-spanning segments of mel permease alters its cationic selectivity properties. The results also indicate that the individual mutations diversely affect mel permease-coupling properties. For example, only permease with Asn in place of Asp-31 or Cys in place of Asp-51 retains the capacity to actively transport melibiose. On the other hand, replacing Asp-55 by Cys produces uncoupling of cosubstrate flows by the carrier but does not hamper sugar translocation. These and other features of the mutant permeases are used to discuss the relative participation of Asp-31, Asp-51, Asp-55, or Asp-120 to the mel symport mechanism and to its ionic selectivity and also the existence of a possible gating mechanism that may contribute the obligatory coupling of cosubstrate flows by the symporter.
Zani
M L
ML
Laboratoire J. Maetz, Département de Biologie Cellulaire et Moléculaire du Commissariat à l'Energie Atomique, Villefranche sur mer, France.
Pourcher
T
T
Leblanc
G
G
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D002412:Cations; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008553:Melibiose / Q000378:metabolism / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D027981:Symporters; D013997:Time Factors
D002412:Cations; D026901:Membrane Transport Proteins; D027981:Symporters; D001224:Aspartic Acid; C021548:melibiose permease; D008553:Melibiose