Purification and characterization of the membrane-associated components 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. The three membrane-associated components of the transport system, the MalK, MalF, and MalG proteins, have been solubilized from the membrane and maltose transport activity has been reconstituted in proteoliposome vesicles (Davidson, A. L., and Nikaido, H. (1990) J. Biol. Chem. 265, 4254-4260). A modification of the reconstitution technique is presented which permits reconstitution from the detergent dodecyl maltoside. Utilizing reconstitution of maltose transport as an assay, we have purified these proteins in the presence of n-dodecyl-beta-D-maltoside. The purified proteins catalyze both maltose transport activity and ATP hydrolysis. In all experiments, the MalF, MalG, and MalK proteins behaved as a multiprotein complex; all three proteins were immunoprecipitated using antibody prepared against MalF, and they copurified, eluting from a gel filtration column between markers of Mr 160,000 and 200,000. Each complex contains two MalK, one MalF, and one MalG proteins, providing two putative sites for ATP hydrolysis. Chemical cross-linking detected specific interactions between MalF and MalG and between MalF and MalK.
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 / Q000302:isolation & purification*; D002352:Carrier Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000032:analysis*; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000302:isolation & purification*; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins; D046912:Multiprotein Complexes; D011233:Precipitin Tests
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; 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; D046912:Multiprotein Complexes; C461599:maltose transport system, E coli; D008320:Maltose; D000255:Adenosine Triphosphate