Overproduction, solubilization, and reconstitution of the maltose transport system from Escherichia coli.
Maltose is transported across the cytoplasmic membrane of Escherichia coli by a binding protein-dependent transport system. We observed a 10-fold increase in the level of transport activity in assays with membrane vesicles when the three membrane-associated components of the transport system (the MalF, MalG, and MalK proteins) were overproduced. In addition, we have successfully reconstituted maltose transport activity in proteoliposome vesicles from solubilized proteins using a detergent dilution procedure. The addition of ATP as an energy source was sufficient to obtain transport, and this activity was dependent on the presence of maltose binding protein and was not seen in proteoliposomes prepared from a strain with a deletion of the maltose genes. We determined that hydrolysis of ATP was directly coupled to maltose uptake. In the majority of these experiments, an average of 1.4 mol of ATP was hydrolyzed for each mole of maltose accumulated. However, in the remaining experiments, ATP hydrolysis was observed to be much higher and averaged 17 mol of ATP hydrolyzed per mol of maltose transported. Possible explanations for a variable stoichiometry are discussed. These results provide strong evidence that it is the hydrolysis of ATP by a component of the transport complex that provides the energy required for active maltose transport.
Davidson
A L
AL
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Nikaido
H
H
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D008081:Liposomes / Q000378:metabolism*; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D010957:Plasmids; D012995:Solubility
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008081:Liposomes; C079123:MalG protein, E coli; C068021:MalK protein, Bacteria; C489398:MalK protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; C461599:maltose transport system, E coli; D008320:Maltose; D000255:Adenosine Triphosphate; D004262:DNA Restriction Enzymes