Functional symmetry of UhpT, the sugar phosphate transporter of Escherichia coli

Mon Chou Fann, Peter C. Maloney

Research output: Contribution to journalArticle

Abstract

UhpT, the sugar phosphate transporter of Escherichia coli, acts to exchange internal inorganic phosphate for external hexose 6-phosphate. Because of this operational asymmetry, we studied variants in which rightside-out (RSO) or inside-out (ISO) orientations could be analyzed independently to ask whether the inward- and outward-facing UhpT surfaces have different substrate specificities. To study the RSO orientation, we constructed a histidine-tagged derivative, His10K291C/K294N, in which the sole external tryptic cleavage site (Lys294) had been removed. Functional assay as well as immunoblot analysis showed that trypsin treatment of proteoliposomes containing His10K291C/K294N led to loss of about 50% of the original population, reflecting retention of only the RSO population. To study the ISO orientation, we used a His10 V284C derivative, in which a newly inserted external cysteine (Cys284) conferred sensitivity to the thiol-reactive agent, 3-(N-maleimidylpropionyl)biocytin. In this case, 3-(N- maleimidylpropionyl)biocytin treatment of proteoliposomes containing His10 V284C gave about a 60% loss of activity, and immunoderection of biotin showed parallel modification of an equivalent fraction of the original population. Together, such findings indicate that the UhpT RSO and ISO orientations are in about equal proportion in proteoliposomes and that a single population can be generated by exposure of these derivatives to the appropriate agent. This allowed us to study proteoliposomes with UhpT functioning in RSO orientation (HiS10K291C/K294N) or ISO orientation (His10 V284C) with respect to the kinetics of glucose 6-phosphate transport by phosphate-loaded proteoliposomes and also the inhibitions found with 2-deoxy-glucose 6-phosphate, mannose 6- phosphate, galactose 6-phosphate, fructose 6-phosphate, and inorganic phosphate. We found no significant differences in the behavior of UhpT in its different orientations, indicating that the transporter possesses an overall functional symmetry.

Original languageEnglish (US)
Pages (from-to)33735-33740
Number of pages6
JournalJournal of Biological Chemistry
Volume273
Issue number50
DOIs
StatePublished - Dec 11 1998

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Phosphate Transport Proteins
Sugar Phosphates
Escherichia coli
Phosphates
Glucose-6-Phosphate
Derivatives
Population
Facings
Hexoses
Biotin
Substrate Specificity
Histidine
Sulfhydryl Compounds
Trypsin
Cysteine
Assays
proteoliposomes
Kinetics
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Functional symmetry of UhpT, the sugar phosphate transporter of Escherichia coli. / Fann, Mon Chou; Maloney, Peter C.

In: Journal of Biological Chemistry, Vol. 273, No. 50, 11.12.1998, p. 33735-33740.

Research output: Contribution to journalArticle

Fann, Mon Chou ; Maloney, Peter C. / Functional symmetry of UhpT, the sugar phosphate transporter of Escherichia coli. In: Journal of Biological Chemistry. 1998 ; Vol. 273, No. 50. pp. 33735-33740.
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abstract = "UhpT, the sugar phosphate transporter of Escherichia coli, acts to exchange internal inorganic phosphate for external hexose 6-phosphate. Because of this operational asymmetry, we studied variants in which rightside-out (RSO) or inside-out (ISO) orientations could be analyzed independently to ask whether the inward- and outward-facing UhpT surfaces have different substrate specificities. To study the RSO orientation, we constructed a histidine-tagged derivative, His10K291C/K294N, in which the sole external tryptic cleavage site (Lys294) had been removed. Functional assay as well as immunoblot analysis showed that trypsin treatment of proteoliposomes containing His10K291C/K294N led to loss of about 50{\%} of the original population, reflecting retention of only the RSO population. To study the ISO orientation, we used a His10 V284C derivative, in which a newly inserted external cysteine (Cys284) conferred sensitivity to the thiol-reactive agent, 3-(N-maleimidylpropionyl)biocytin. In this case, 3-(N- maleimidylpropionyl)biocytin treatment of proteoliposomes containing His10 V284C gave about a 60{\%} loss of activity, and immunoderection of biotin showed parallel modification of an equivalent fraction of the original population. Together, such findings indicate that the UhpT RSO and ISO orientations are in about equal proportion in proteoliposomes and that a single population can be generated by exposure of these derivatives to the appropriate agent. This allowed us to study proteoliposomes with UhpT functioning in RSO orientation (HiS10K291C/K294N) or ISO orientation (His10 V284C) with respect to the kinetics of glucose 6-phosphate transport by phosphate-loaded proteoliposomes and also the inhibitions found with 2-deoxy-glucose 6-phosphate, mannose 6- phosphate, galactose 6-phosphate, fructose 6-phosphate, and inorganic phosphate. We found no significant differences in the behavior of UhpT in its different orientations, indicating that the transporter possesses an overall functional symmetry.",
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