MUTATION OF A SINGLE MALK SUBUNIT SEVERELY IMPAIRS MALTOSE TRANSPORT ACTIVITY IN ESCHERICHIA-COLI

The maltose transport system of Escherichia call, a member of the ABC transport superfamily of proteins, consists of a periplasmic maltose b inding protein and a membrane-associated translocation complex that co ntains 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, Co mplexes with mutations in a single subunit were obtained by attaching a polyhistidine tag to the mutagenized version of MalK and by coexpres sing both wild-type MalK and mutant (His)(6)MalK in the same cell. Hyb rid complexes containing one mutant (His)(6)MalK subunit and one wild- type MalK subunit were separated from those containing two mutant (His )(6)MalK proteins based on differential affinities for a metal chelate column. Purified transport complexes were reconstituted into proteoli posome vesicles and assayed for maltose transport and ATPase activitie s. When a conserved lysine residue at position 42 that is involved in ATP binding was replaced with asparagine in both MalK subunits, maltos e transport and ATPase activities were reduced to 1% of those of the w ild type. When the mutation was present in only one of the two subunit s, the complex had 6% of the wild-type activities. Replacement of a co nserved histidine residue at position 192 in MalK with arginine genera ted similar results. It is clear from these results that two functiona l MalK proteins are required for transport activity and that the two n ucleotide-binding domains do not function independently to catalyze tr ansport.