Topology of the Escherichia coli uhpT sugar-phosphate transporter analyzed by using TnphoA fusions.
The Escherichia coli uhpT protein catalyzes the active transport of sugar-phosphates by an obligatory exchange mechanism. To examine its transmembrane topology, we isolated a collection of uhpT-phoA fusions encoding hybrid proteins of different lengths from the N terminus of UhpT fused to alkaline phosphatase by using transposon TnphoA. These fusions displayed different levels of alkaline phosphatase activity, although comparable levels of full-length UhpT-PhoA proteins were produced in maxicells of both high- and low-activity fusions. The full-length protein species were unstable and were degraded to the size of the alkaline phosphatase moiety in the case of a high-activity fusion or to small fragments in the case of a low-activity fusion. The enzyme activity present in low-activity fusions appeared to result from export of a small proportion of the fusion proteins to the periplasmic space. Although fusions were not obtained in all predicted extramembranous loops, the deduced topology of UhpT was consistent with a model of 12 membrane-spanning regions oriented with the amino and carboxyl termini in the cytoplasm.
Lloyd
A D
AD
Department of Microbiology, School of Medicine, University of Virginia, Charlottesville 22908.
Kadner
R J
RJ
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004274:DNA, Recombinant / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D015345:Oligonucleotide Probes; D010957:Plasmids; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004251:DNA Transposable Elements; D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins; C063498:UhpT protein, E coli