CHARACTERIZATION OF THE STRUCTURAL REQUIREMENTS FOR ASSEMBLY AND NUCLEOTIDE-BINDING OF AN ATP-BINDING CASSETTE TRANSPORTER - THE MALTOSE TRANSPORT-SYSTEM OF ESCHERICHIA-COLI

The periplasmic maltose-binding protein-dependent, maltose transport s ystem of Escherichia coli is a well studied member of the ATP-binding cassette family of transport ATPases. In addition to the water-soluble maltose-binding protein, the system comprises three membrane proteins , MalF, MalG, and MalK, which form a heterotetrameric complex (FGK2) i n the cytoplasmic membrane. The purified complex exhibits transport-as sociated ATPase activity. To characterize the requirements for nucleot ide binding and hydrolysis by the FGK2 complex, we used plasmids to ex press different combinations of the individual subunits as well as mut ant forms of the MalK subunit. Prior to measuring nucleotide binding, we examined membrane preparations for the presence of each subunit fro m strains that contained all possible permutations of the three struct ural genes, malF, malG, and malk. We found that when all three genes w ere present or when malF and malK were present together, the correspon ding antigens were detected easily on Western immunoblots and were sol uble in the non-ionic detergent, Triton X- 1 00. In contrast, all othe r permutations resulted in decreased amounts of antigen or antigen tha t was Triton X-100-insoluble. We relied on photocross-linking with 8-a zido-[P-32]ATP and ATP hydrolysis as indicators of the ability of the transport complex to interact with purine nucleotides. 8-Azido-[P-32]A TP was photocross-linked to the MalK subunit. Photolabeling of MalK wa s inhibited by ATP, ADP, and GTP and not by other nucleotides. Photola beling of MalK required the presence of MalF but not MalG. Mutations i n malk that affect amino acid residues thought to be directly involved in nucleotide binding did indeed abolish labeling and resulted in los s of transport activity without affecting protein stability. In genera l, ATP hydrolysis correlated with the photocross-linking. A notable ex ception is the MalK941 mutant protein which retained the ability to be labeled by 8-azido-[P-32]ATP but was unable to catalyze detectable le vels of ATP hydrolysis. Some, but not all, of the malK mutations were dominant to wild type. To study the mechanism of dominance we devised a means of measuring the ability of different wild-type and mutant Mal K proteins to interact with the MalF and MalG subunits. This assay rel ies on the fact that, when a bifunctional MalK-LacZ hybrid protein is associated with the MalF and MalG subunits, it is membrane-bound. Exce ss MalK competed with the MalK-LacZ hybrid protein for sites in the me mbrane and resulted in the hybrid fractionating as a soluble protein. We found that the mutant MalK proteins encoded by the strongly dominan t malk mutations competed with the MalK-LacZ protein as efficiently as wild type. In contrast, the recessive malK mutations encoded MalK mut ant proteins that did not compete with the hybrid, indicating that the y were partially defective in the ability to interact with either MalF and/or MalG.