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 system 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) in the cytoplasmic membrane. The purified complex exhibits transport-associated ATPase activity. To characterize the requirements for nucleotide binding and hydrolysis by the FGK2 complex, we used plasmids to express different combinations of the individual subunits as well as mutant forms of the MalK subunit. Prior to measuring nucleotide binding, we examined membrane preparations for the presence of each subunit from strains that contained all possible permutations of the three structural genes, malF, malG, and malK. We found that when all three genes were present or when malF and malK were present together, the corresponding antigens were detected easily on Western immunoblots and were soluble in the non-ionic detergent, Triton X-100. In contrast, all other permutations resulted in decreased amounts of antigen or antigen that was Triton X-100-insoluble. We relied on photocross-linking with 8-azido-[32P]ATP and ATP hydrolysis as indicators of the ability of the transport complex to interact with purine nucleotides. 8-Azido-[32P]ATP was photocross-linked to the MalK subunit. Photolabeling of MalK was inhibited by ATP, ADP, and GTP and not by other nucleotides. Photolabeling of MalK required the presence of MalF but not MalG. Mutations in malK that affect amino acid residues thought to be directly involved in nucleotide binding did indeed abolish labeling and resulted in loss of transport activity without affecting protein stability. In general, ATP hydrolysis correlated with the photocross-linking. A notable exception is the MalK941 mutant protein which retained the ability to be labeled by 8-azido-[32P]ATP but was unable to catalyze detectable levels 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 MalK proteins to interact with the MalF and MalG subunits. This assay relies on the fact that, when a bifunctional MalK-LacZ hybrid protein is associated with the MalF and MalG subunits, it is membrane-bound. Excess MalK competed with the MalK-LacZ hybrid protein for sites in the membrane and resulted in the hybrid fractionating as a soluble protein.(ABSTRACT TRUNCATED AT 400 WORDS)
Panagiotidis
C H
CH
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Reyes
M
M
Sievertsen
A
A
Boos
W
W
Shuman
H A
HA
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000378:metabolism*; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D017931:DNA Primers; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; 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; D011994:Recombinant Proteins; C461599:maltose transport system, E coli; D008320:Maltose; D000255:Adenosine Triphosphate