Mutation of a single MalK subunit severely impairs maltose transport activity in Escherichia coli.
The maltose transport system of Escherichia coli, a member of the ABC transport superfamily of proteins, consists of a periplasmic maltose binding protein and a membrane-associated translocation complex that contains two copies of the ATP-binding protein MalK. To examine the need for two nucleotide-binding domains in this transport complex, one of the two MalK subunits was inactivated by site-directed mutagenesis. Complexes with mutations in a single subunit were obtained by attaching a polyhistidine tag to the mutagenized version of MalK and by coexpressing both wild-type MalK and mutant (His)6MalK in the same cell. Hybrid complexes containing one mutant (His)6MalK subunit and one wild-type MalK subunit were separated from those containing two mutant (His)6MalK proteins based on differential affinities for a metal chelate column. Purified transport complexes were reconstituted into proteoliposome vesicles and assayed for maltose transport and ATPase activities. When a conserved lysine residue at position 42 that is involved in ATP binding was replaced with asparagine in both MalK subunits, maltose transport and ATPase activities were reduced to 1% of those of the wild type. When the mutation was present in only one of the two subunits, the complex had 6% of the wild-type activities. Replacement of a conserved histidine residue at position 192 in MalK with arginine generated similar results. It is clear from these results that two functional MalK proteins are required for transport activity and that the two nucleotide-binding domains do not function independently to catalyze transport.
Davidson
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Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030, USA. davidson@bcm.tmc.edu
Sharma
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D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006639:Histidine / Q000502:physiology; D008239:Lysine / Q000502:physiology; D008320:Maltose / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D033902:Periplasmic Binding Proteins; D011510:Proteolipids / Q000378:metabolism; D011993:Recombinant Fusion Proteins
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; C079123:MalG protein, E coli; C068021:MalK protein, Bacteria; C489398:MalK protein, E coli; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011510:Proteolipids; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; C020485:proteoliposomes; D006639:Histidine; D008320:Maltose; D000251:Adenosine Triphosphatases; D008239:Lysine