Construction of a functional lactose permease devoid of cysteine residues.
By use of oligonucleotide-directed, site-specific mutagenesis, a lactose (lac) permease molecule was constructed in which all eight cysteinyl residues were simultaneously mutagenized (C-less permease). Cys154 was replaced with valine, and Cys117, -148, -176, -234, -333, -353, and -355 were replaced with serine. Remarkably, C-less permease catalyzes lactose accumulation in the presence of a transmembrane proton electrochemical gradient (interior negative and alkaline). Thus, in intact cells and right-side-out membrane vesicles containing comparable amounts of wild-type and Cys-less permease, the mutant protein catalyzes lactose transport at a maximum velocity and to a steady-state level of accumulation of about 35% and 55%, respectively, of wild-type with a similar apparent Km (ca. 0.3 mM). As anticipated, moreover, active lactose transport via C-less permease is completely resistant to inactivation by N-ethylmaleimide. Finally, C-less permease also catalyzes efflux and equilibrium exchange at about 35% of wild-type activity. The results provide definitive evidence that sulfhydryl groups do not play an essential role in the mechanism of lactose/H+ symport. Potential applications of the C-less mutant to studies of static and dynamic aspects of permease structure/function are discussed.
van Iwaarden
P R
PR
Howard Hughes Medical Institute, Department of Physiology, Los Angeles, California 90024-1570.
Pastore
J C
JC
Konings
W N
WN
Kaback
H R
HR
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000235:genetics*; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D007785:Lactose; D003545:Cysteine