Characterization of Escherichia coli lactose carrier mutants that transport protons without a cosubstrate: Probes for the energy barrier to uncoupled transport

Steven King, T. Hastings Wilson

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

The Escherichia coli lactose carrier is an energy-transducing H+/galactoside cotransport protein which strictly couples sugar and proton transport in 1:1 stoichiometry. Here we describe five lactose carrier mutants which catalyze "uncoupled" sugar-independent H+ transport. Symptoms similar to uncoupling by a proton ionophore have been observed in cells expressing these mutant carriers. The mutations occur at two separate loci, encoding substitutions either for alanine 177 (valine) or tyrosine 236 (histidine, asparagine, phenylalanine, or serine). Compared to the parent, cells expressing the valine 177 carrier grew slowly on minimal media with glucose as carbon source. When washed cells were incubated in the absence of added sugars the mutant showed a reduced protonmotive force compared with the parent. Addition of either thiodigalactoside or α-p-nitrophenylgalactoside reduced the defect in protonmotive force. Sugar-independent H+ entry rate into cells expressing either the normal carrier or the Val-177 mutant were measured directly using the pH electrode. Following sudden acidification of the external medium (by either oxygen-pulse or acid-pulse) protons entered more rapidly into cells expressing the Val-177 carrier. This novel sugar-independent mode of H+ transport probably depends on an acquired capacity of the Val-177 carrier to bind the transported proton with higher than normal affinity in a transition state involving the binary carrier/H+ complex.

Original languageEnglish (US)
Pages (from-to)9645-9651
Number of pages7
JournalJournal of Biological Chemistry
Volume265
Issue number17
StatePublished - Jun 15 1990
Externally publishedYes

Fingerprint

Energy barriers
Lactose
Sugars
Escherichia coli
Protons
Cells
Valine
Proton Ionophores
Nitrophenylgalactosides
Galactosides
Acidification
Asparagine
Phenylalanine
Histidine
Stoichiometry
Alanine
Serine
Tyrosine
Electrodes
Substitution reactions

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{b1ff7db654e249e6bfa7c71008a81b7c,
title = "Characterization of Escherichia coli lactose carrier mutants that transport protons without a cosubstrate: Probes for the energy barrier to uncoupled transport",
abstract = "The Escherichia coli lactose carrier is an energy-transducing H+/galactoside cotransport protein which strictly couples sugar and proton transport in 1:1 stoichiometry. Here we describe five lactose carrier mutants which catalyze {"}uncoupled{"} sugar-independent H+ transport. Symptoms similar to uncoupling by a proton ionophore have been observed in cells expressing these mutant carriers. The mutations occur at two separate loci, encoding substitutions either for alanine 177 (valine) or tyrosine 236 (histidine, asparagine, phenylalanine, or serine). Compared to the parent, cells expressing the valine 177 carrier grew slowly on minimal media with glucose as carbon source. When washed cells were incubated in the absence of added sugars the mutant showed a reduced protonmotive force compared with the parent. Addition of either thiodigalactoside or α-p-nitrophenylgalactoside reduced the defect in protonmotive force. Sugar-independent H+ entry rate into cells expressing either the normal carrier or the Val-177 mutant were measured directly using the pH electrode. Following sudden acidification of the external medium (by either oxygen-pulse or acid-pulse) protons entered more rapidly into cells expressing the Val-177 carrier. This novel sugar-independent mode of H+ transport probably depends on an acquired capacity of the Val-177 carrier to bind the transported proton with higher than normal affinity in a transition state involving the binary carrier/H+ complex.",
author = "Steven King and Wilson, {T. Hastings}",
year = "1990",
month = "6",
day = "15",
language = "English (US)",
volume = "265",
pages = "9645--9651",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "17",

}

TY - JOUR

T1 - Characterization of Escherichia coli lactose carrier mutants that transport protons without a cosubstrate

T2 - Probes for the energy barrier to uncoupled transport

AU - King, Steven

AU - Wilson, T. Hastings

PY - 1990/6/15

Y1 - 1990/6/15

N2 - The Escherichia coli lactose carrier is an energy-transducing H+/galactoside cotransport protein which strictly couples sugar and proton transport in 1:1 stoichiometry. Here we describe five lactose carrier mutants which catalyze "uncoupled" sugar-independent H+ transport. Symptoms similar to uncoupling by a proton ionophore have been observed in cells expressing these mutant carriers. The mutations occur at two separate loci, encoding substitutions either for alanine 177 (valine) or tyrosine 236 (histidine, asparagine, phenylalanine, or serine). Compared to the parent, cells expressing the valine 177 carrier grew slowly on minimal media with glucose as carbon source. When washed cells were incubated in the absence of added sugars the mutant showed a reduced protonmotive force compared with the parent. Addition of either thiodigalactoside or α-p-nitrophenylgalactoside reduced the defect in protonmotive force. Sugar-independent H+ entry rate into cells expressing either the normal carrier or the Val-177 mutant were measured directly using the pH electrode. Following sudden acidification of the external medium (by either oxygen-pulse or acid-pulse) protons entered more rapidly into cells expressing the Val-177 carrier. This novel sugar-independent mode of H+ transport probably depends on an acquired capacity of the Val-177 carrier to bind the transported proton with higher than normal affinity in a transition state involving the binary carrier/H+ complex.

AB - The Escherichia coli lactose carrier is an energy-transducing H+/galactoside cotransport protein which strictly couples sugar and proton transport in 1:1 stoichiometry. Here we describe five lactose carrier mutants which catalyze "uncoupled" sugar-independent H+ transport. Symptoms similar to uncoupling by a proton ionophore have been observed in cells expressing these mutant carriers. The mutations occur at two separate loci, encoding substitutions either for alanine 177 (valine) or tyrosine 236 (histidine, asparagine, phenylalanine, or serine). Compared to the parent, cells expressing the valine 177 carrier grew slowly on minimal media with glucose as carbon source. When washed cells were incubated in the absence of added sugars the mutant showed a reduced protonmotive force compared with the parent. Addition of either thiodigalactoside or α-p-nitrophenylgalactoside reduced the defect in protonmotive force. Sugar-independent H+ entry rate into cells expressing either the normal carrier or the Val-177 mutant were measured directly using the pH electrode. Following sudden acidification of the external medium (by either oxygen-pulse or acid-pulse) protons entered more rapidly into cells expressing the Val-177 carrier. This novel sugar-independent mode of H+ transport probably depends on an acquired capacity of the Val-177 carrier to bind the transported proton with higher than normal affinity in a transition state involving the binary carrier/H+ complex.

UR - http://www.scopus.com/inward/record.url?scp=0025290251&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025290251&partnerID=8YFLogxK

M3 - Article

C2 - 2161839

AN - SCOPUS:0025290251

VL - 265

SP - 9645

EP - 9651

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 17

ER -