Direct evidence for substrate-induced proton release in detergent-solubilized EmrE, a multidrug transporter.
A novel approach to study coupling of substrate and ion fluxes is presented. EmrE is an H(+)-coupled multidrug transporter from Escherichia coli. Detergent-solubilized EmrE binds substrate with high affinity in a pH-dependent mode. Here we show, for the first time in an ion-coupled transporter, substrate-induced release of protons in a detergent-solubilized preparation. The direct measurements allow for an important quantitation of the phenomenon. Thus, stoichiometry of the release in the wild type and a mutant with a single carboxyl at position 14 is very similar and about 0.8 protons/monomer. The findings demonstrate that the only residue involved in proton release is a highly conserved membrane-embedded glutamate (Glu-14) and that all the Glu-14 residues in the EmrE functional oligomer participate in proton release. Furthermore, from the pH dependence of the release we determined the pK of Glu-14 as 8.5 and for an aspartate replacement at the same position as 6.7. The high pK of the carboxyl at position 14 is essential for coupling of fluxes of protons and substrates.
Soskine
Misha
M
Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel.
Adam
Yoav
Y
Schuldiner
Shimon
S
D017920:Antiporters / Q000737:chemistry* / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D018698:Glutamic Acid / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008954:Models, Biological; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011522:Protons
D017920:Antiporters; D003902:Detergents; D029968:Escherichia coli Proteins; D007477:Ions; D008565:Membrane Proteins; D011522:Protons; C075436:EmrE protein, E coli; D001224:Aspartic Acid; D018698:Glutamic Acid; D004024:Dicyclohexylcarbodiimide; D006859:Hydrogen