Characterization of purified, reconstituted site-directed cysteine mutants of the lactose permease of Escherichia coli.
lac permease mutated at each of the 8 cysteinyl residues in the molecule was solubilized from the membrane, purified, and reconstituted into proteoliposomes. The transport activity of proteoliposomes reconstituted with each mutant permease relative to the wild-type is virtually identical with that reported for intact cells and/or right-side-out membrane vesicles. Moreover, a double mutant containing Ser in place of both Cys148 and Cys154 exhibits significant ability to catalyze active lactose transport. The results provide strong confirmation for the contention that cysteinyl residues in lac permease do not play an important role in the transport mechanism. The effect of sulfhydryl oxidant 5-hydroxy-2-methyl-1,4-naphthoquinone on lactose transport in proteoliposomes reconstituted with wild-type or mutant permeases was also investigated, and the results indicate that inactivation is probably due to formation of a covalent adduct with Cys148 and/or Cys154 rather than disulfide formation. Thus, it seems unlikely that sulfhydryl-disulfide interconversion functions to regulate permease activity.
van Iwaarden
P R
PR
Department of Microbiology, University of Groningen, Haren, The Netherlands.
Driessen
A J
AJ
Menick
D R
DR
Kaback
H R
HR
Konings
W N
WN
D000595:Amino Acid Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D003545:Cysteine / Q000737:chemistry*; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007785:Lactose / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D009285:Naphthoquinones / Q000494:pharmacology; D010957:Plasmids; D011510:Proteolipids / Q000378:metabolism; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D009285:Naphthoquinones; D011510:Proteolipids; D027981:Symporters; C020485:proteoliposomes; C021499:lactose permease; D007785:Lactose; D003545:Cysteine; D004229:Dithiothreitol; C014758:plumbagin