UhpT, the sugar phosphate antiporter of Escherichia coli, functions as a monomer

Suresh V. Ambudkar, Vellareddy Anantharam, Peter C. Maloney

Research output: Contribution to journalArticle

Abstract

We have characterized the minimal functioning unit of UhpT, the secondary carrier that mediates exchange of phosphate and glucose 6-phosphate in Escherichia coli. Membranes of a UhpT overproducing strain were solubilized with 1.25% octyl β-D-glucopyranoside, in the presence of 0.1% E. coli phospholipid and with 20% glycerol as the osmolyte stabilant. That soluble UhpT could bind its natural substrates was indicated by the protections afforded by sugar phosphates against thermal inactivation or chemical modification with pyridoxal 5′-phosphate. Moreover, the degree of protection correlated with the strength of interaction between UhpT and the test substrate (2-deoxyglucose 6-phosphate = glucose 6-phosphate > galactose 6-phosphate = glucose 1-phosphate ≫ glucose 6-sulfate). Other experiments demonstrated that soluble UhpT existed as a monomer. For example, during both high performance liquid chromatography and conventional gel permeation chromatography, the elution pattern of UhpT activity was measured directly by a rapid reconstitution technique. In both cases, and in the presence and absence of substrate, UhpT activity traveled as a single component of Mr 53,000, corresponding closely to the sequence prediction of 50,600. Finally, reconstitution was studied at protein to lipid ratios low enough to achieve between 0.075 and 1.5 UhpT monomers/proteoliposome. Specific activity was constant throughout this range, a finding consistent with the idea of a functional monomer. Mitochondria and chloroplasts provide the only other anion exchange carriers described at this level of biochemical resolution, and these organelle antiporters function as dimers. By contrast, work summarized here places their bacterial counterpart, UhpT, in the same class as the lactose carrier of E. coli and the glucose carrier of the human erythrocyte, both of which function as monomers. Consideration of this pattern in conjunction with the known hydropathy profiles of these proteins suggests a novel scheme for the classification of all secondary carriers, with implications for both the structure and origin of these transport proteins.

Original languageEnglish (US)
Pages (from-to)12287-12292
Number of pages6
JournalJournal of Biological Chemistry
Volume265
Issue number21
StatePublished - Jul 25 1990

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Sugar Phosphates
Antiporters
Escherichia coli
Glucose-6-Phosphate
Monomers
Pyridoxal Phosphate
Substrates
Chloroplasts
Lactose
Glucose
Workplace
Organelles
Glycerol
Mitochondria
Gel Chromatography
Anions
Phospholipids
Carrier Proteins
Chemical modification
High performance liquid chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ambudkar, S. V., Anantharam, V., & Maloney, P. C. (1990). UhpT, the sugar phosphate antiporter of Escherichia coli, functions as a monomer. Journal of Biological Chemistry, 265(21), 12287-12292.

UhpT, the sugar phosphate antiporter of Escherichia coli, functions as a monomer. / Ambudkar, Suresh V.; Anantharam, Vellareddy; Maloney, Peter C.

In: Journal of Biological Chemistry, Vol. 265, No. 21, 25.07.1990, p. 12287-12292.

Research output: Contribution to journalArticle

Ambudkar, SV, Anantharam, V & Maloney, PC 1990, 'UhpT, the sugar phosphate antiporter of Escherichia coli, functions as a monomer', Journal of Biological Chemistry, vol. 265, no. 21, pp. 12287-12292.
Ambudkar SV, Anantharam V, Maloney PC. UhpT, the sugar phosphate antiporter of Escherichia coli, functions as a monomer. Journal of Biological Chemistry. 1990 Jul 25;265(21):12287-12292.
Ambudkar, Suresh V. ; Anantharam, Vellareddy ; Maloney, Peter C. / UhpT, the sugar phosphate antiporter of Escherichia coli, functions as a monomer. In: Journal of Biological Chemistry. 1990 ; Vol. 265, No. 21. pp. 12287-12292.
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