Abstract
Maltose transport across the cytoplasmic membrane of Escherichia coli is catalyzed by a periplasmic binding protein-dependent transport system and energized by ATP. The maltose system, a member of the ATP-binding cassette or ABC transport family, contains two copies of an ATP-binding protein in a complex with two integral membrane proteins. ATP hydrolysis by the transport complex can be assayed following reconstitution into proteoliposomes in the presence of maltose binding protein and maltose. Mutations in the transport complex that permit binding protein-independent transport render ATP hydrolysis constitutive so that hydrolysis can also be assayed with the transport complex in detergent solution. We have used both of these systems to study the role of two ATP binding sites in ATP hydrolysis. We found that both the wild-type and the binding protein-independent systems hydrolyzed ATP with positive cooperativity, suggesting that the two ATP binding sites interact. Vanadate inhibited the ATPase activity of the transport complex with 50% inhibition occurring at 10 mum vanadate. In detergent solution, the degree of cooperativity in the binding protein-independent complex decreased with increasing pH. The loss of cooperativity was accompanied by a decrease in ATPase activity and a decrease in sensitivity to vanadate. Because reconstitution of the complex into a lipid bilayer prevented the loss of cooperativity, we expect that ATP hydrolysis is cooperative in vivo. The mutations leading to binding protein-independent transport do not significantly alter the affinity, cooperativity, vanadate sensitivity, or substrate specificity of the ATP binding sites during hydrolysis. These results justify the use of the binding protein-independent system to investigate the mechanism of transport and hydrolysis.
Highlights
EXPERIMENTAL PROCEDURESStrains and Plasmids—Strain HN741 (E. coli K-12 argH his rpsL1 malTc ⌬malB13 ⌬atpBC ilv::Tn10/FЈ lacIq Tn5) [11] containing chromosomal deletions of the genes encoding the maltose transport system
In proteoliposomes, MalFGK2 requires both maltose and maltose binding protein (MBP) to hydrolyze ATP [11]
PH Dependence of ATP Hydrolysis—ATP hydrolysis was assayed as a function of pH using either the wild-type transport complex or a transport complex generated from the malF500 allele, which had the highest constitutive ATPase activity of the binding protein-independent mutants tested [11]
Summary
Strains and Plasmids—Strain HN741 (E. coli K-12 argH his rpsL1 malTc ⌬malB13 ⌬atpBC ilv::Tn10/FЈ lacIq Tn5) [11] containing chromosomal deletions of the genes encoding the maltose transport system. Vesicles were resuspended in a solution of 20 mM Tris-HCl, pH 8, 5 mM MgCl2, 1 mM dithiothreitol, and 20% glycerol, and proteins were solubilized by the addition of the detergents octyl glucoside or dodecyl maltoside to final concentrations of 1%. The ability of the ATPase to hydrolyze other nucleotides was tested as described above using sodium acetate buffer at pH 5 for the detergent-soluble form of the enzyme and Bis-Tris at pH 6 for the reconstituted systems, except that unlabeled substrates were employed and Pi was determined using a microtiter plate method [17]. Protein concentrations were determined as described previously [9] using the method of Schaffner and Weissmann [18]
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