Properties of a LacY efflux mutant.
Crystal structures of lactose permease from Escherichia coli (LacY) exhibit two six-helix bundles with 2-fold pseudosymmetry separated by a large hydrophilic cavity. The cavity is open only on the cytoplasmic side and contains the side chains important for both sugar and H(+) binding at the apex in the middle of the protein; the periplasmic side is tightly closed. A plethora of biochemical and biophysical data strongly support an alternating access mechanism in which both the sugar- and H(+)-binding sites are exposed alternatively to either side of the membrane by reciprocal opening and closing of cytoplasmic and periplasmic cavities. Here we describe a unique mutation that results in an increase in sugar efflux. Asp240 (helix VII), which interacts with Lys319 (helix X), also comprises part of a salt-bridge/H-bond network that is critically involved in the mechanism of sugar/H(+) symport. The mutant, which contains Glu in place of Asp240, exhibits a marked decrease in active lactose transport and an enhanced rate of downhill lactose/H(+) efflux. Transport is increased to normal levels when the sugar concentration is increased 10-fold, consistent with the decrease in sugar affinity observed for this mutant. Taken as a whole, the results suggest that the primary defect induced by the mutation may involve a decrease in affinity for H(+).
Guan
Lan
L
Department of Physiology, University of California Los Angeles, Los Angeles, California 90095-1662, USA.
Kaback
H Ronald
HR
D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001693:Biological Transport, Active / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics; D006859:Hydrogen / Q000378:metabolism; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D021381:Protein Transport / Q000235:genetics
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D006859:Hydrogen; C021499:lactose permease; D007785:Lactose