Purification and characterization of an outer membrane-bound protein involved in long-chain fatty acid transport in Escherichia coli

Paul N Black, B. Said, C. R. Ghosn, J. V. Beach, W. D. Nunn

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Abstract

We report the purification and localization of the fadL gene product (FLP), an essential component of the long-chain fatty acid transport machinery in Escherichia coli. FLP was extracted from total membranes by differential extraction with the nonionic detergents Tween 20 and Triton X-100. This protein was further purified from a Tween 20-insoluble-Triton X-100-soluble extract by salt fractionation, gel filtration chromatography, and hydrophobic interaction chromatography. This regime results in a 95-fold purification of FLP from total membranes. The purified protein preparation was homogeneous based on silver staining and gave the characteristic behavior established for the fadL gene product in the presence of sodium dodecyl sulfate at different temperatures prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (M(r) of 33,000 when heated at 25°C and M(r) of 43,000 when heated at 100°C) and on two-dimensional polyacrylamide gels (pI of 4.6 and a M(r) of 33,000). Purified FLP was rich in hydrophobic residues accounting for approximately 45% of the total amino acid composition. To localize FLP, antisera were raised against the purified protein and were used to probe differentially fractionated membranes by Western immunoblotting. This procedure demonstrated the presence of this protein only in the outer membrane fraction of fadL+ strains. We confirmed the outer membrane localization of FLP by measuring long-chain fatty acid transport in fadL+ and fadL strains treated with EDTA to alter outer membrane permeability and in spheroplasts generated from fadL+ and fadL strains. Both EDTA-treated cells and spheroplasts transportd long-chain fatty acids at essentially the same rate regardless of whether they contained a wild-type or mutant fadL gene. These data imply that FLP is a protein in the outer membrane which is specifically involved in long-chain fatty acid transport.

Original languageEnglish (US)
Pages (from-to)1412-1419
Number of pages8
JournalJournal of Biological Chemistry
Volume262
Issue number3
StatePublished - May 4 1987

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Escherichia coli
Purification
Membrane Proteins
Fatty Acids
Genes
Membranes
Proteins
Spheroplasts
Polysorbates
Octoxynol
Edetic Acid
Sodium Dodecyl Sulfate
Chromatography
Silver Staining
Essential Genes
Hydrophobic and Hydrophilic Interactions
Detergents
Fractionation
Electrophoresis
Gel Chromatography

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Purification and characterization of an outer membrane-bound protein involved in long-chain fatty acid transport in Escherichia coli. / Black, Paul N; Said, B.; Ghosn, C. R.; Beach, J. V.; Nunn, W. D.

In: Journal of Biological Chemistry, Vol. 262, No. 3, 04.05.1987, p. 1412-1419.

Research output: Contribution to journalArticle

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abstract = "We report the purification and localization of the fadL gene product (FLP), an essential component of the long-chain fatty acid transport machinery in Escherichia coli. FLP was extracted from total membranes by differential extraction with the nonionic detergents Tween 20 and Triton X-100. This protein was further purified from a Tween 20-insoluble-Triton X-100-soluble extract by salt fractionation, gel filtration chromatography, and hydrophobic interaction chromatography. This regime results in a 95-fold purification of FLP from total membranes. The purified protein preparation was homogeneous based on silver staining and gave the characteristic behavior established for the fadL gene product in the presence of sodium dodecyl sulfate at different temperatures prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (M(r) of 33,000 when heated at 25°C and M(r) of 43,000 when heated at 100°C) and on two-dimensional polyacrylamide gels (pI of 4.6 and a M(r) of 33,000). Purified FLP was rich in hydrophobic residues accounting for approximately 45{\%} of the total amino acid composition. To localize FLP, antisera were raised against the purified protein and were used to probe differentially fractionated membranes by Western immunoblotting. This procedure demonstrated the presence of this protein only in the outer membrane fraction of fadL+ strains. We confirmed the outer membrane localization of FLP by measuring long-chain fatty acid transport in fadL+ and fadL strains treated with EDTA to alter outer membrane permeability and in spheroplasts generated from fadL+ and fadL strains. Both EDTA-treated cells and spheroplasts transportd long-chain fatty acids at essentially the same rate regardless of whether they contained a wild-type or mutant fadL gene. These data imply that FLP is a protein in the outer membrane which is specifically involved in long-chain fatty acid transport.",
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