Active transport of maltose in membrane vesicles obtained from Escherichia coli cells producing tethered maltose-binding protein.
Attempts to reconstitute periplasmic binding protein-dependent transport activity in membrane vesicles have often resulted in systems with poor and rather inconsistent activity, possibly because of the need to add a large excess of purified binding protein to the vesicles. We circumvented this difficulty by using a mutant which produces a precursor maltose-binding protein that is translocated across the cytoplasmic membrane but is not cleaved by the signal peptidase (J. D. Fikes and P. J. Bassford, Jr., J. Bacteriol. 169:2352-2359, 1987). The protein remains tethered to the cytoplasmic membrane, presumably through the hydrophobic signal sequence, and we show here that the spheroplasts and membrane vesicles prepared from this mutant catalyze active maltose transport without the addition of purified maltose-binding protein. In vesicles, the transport requires electron donors, such as ascorbate and phenazine methosulfate or D-lactate. However, inhibition by dicyclohexylcarbodiimide and stimulation of transport by the inculsion of ADP or ATP in the intravesicular space suggest that ATP (or compounds derived from it) is involved in the energization of the transport. The transport activity of intact cells can be recovered without much inactivation in the vesicles, and their high activity and ease of preparation will be useful in studies of the mechanism of the binding protein-dependent transport process.
Dean
D A
DA
Department of Microbiology and Immunology, University of California, Berkeley 94720.
Fikes
J D
JD
Gehring
K
K
Bassford
P J
PJ
Nikaido
H
H
D018528:ATP-Binding Cassette Transporters; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000096:biosynthesis*; D002462:Cell Membrane / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008320:Maltose / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000096:biosynthesis*; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D010957:Plasmids; D013104:Spheroplasts / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C461599:maltose transport system, E coli; D008320:Maltose