Escherichia coli TonB protein is exported from the cytoplasm without proteolytic cleavage of its amino terminus

Kathleen Postle, J. T. Skare

Research output: Contribution to journalArticle

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Abstract

The requirement for TonB protein in a variety of membrane-related processes suggests that TonB is an envelope protein. Consistent with this suggestion, the deduced TonB amino acid sequence (Postle, K., and Good R. F., (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 5235-5239) contains an amino-terminal region similar to leader (signal) sequences of exported proteins, although its charged region falls outside the rules which characterize these sequences (von Heijne, G. (1985) J. Mol. Biol. 184, 99-105). The deduced TonB amino acid sequence contains three potential methionine start codons in the first six codons of the open reading frame. In this report, we show, by Edman degradation of [35S]methionine-labeled protein, that TonB protein synthesized in vitro initiates at the third of these methionine codons. A method for detecting TonB synthesized in vivo has been developed that involves expression of TonB from the λP(L) promoter and pulse labeling with [35S]methionine. TonB synthesized in vivo has a chemical half-life of 10 min at 42°C. It is exported from the cytoplasm, as determined by proteinase K accessibility experiments. It fractionates with spheroplasts under conditions where maltose-binding protein fractionates with the periplasm. It has the same mobility in three different polyacrylamide gel systems as TonB synthesized in vitro. We conclude that the amino terminus of TonB is uncleaved following its export from the cytoplasm and that TonB is a membrane-associated protein. Characterization of a tonB-phoA gene fusion suggests that the amino-terminal 41 amino acids of TonB are sufficient to promote export of the fusion protein and presumably TonB as well. Models for TonB orientation within the cell envelope are presented.

Original languageEnglish (US)
Pages (from-to)11000-11007
Number of pages8
JournalJournal of Biological Chemistry
Volume263
Issue number22
StatePublished - 1988

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Escherichia coli Proteins
Escherichia coli
Cytoplasm
Methionine
Proteins
Amino Acids
Codon
Amino Acid Sequence
Fusion reactions
Spheroplasts
Maltose-Binding Proteins
Endopeptidase K
Periplasm
Initiator Codon
Gene Fusion
Protein Sorting Signals
Labeling
Open Reading Frames
Half-Life
Membrane Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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abstract = "The requirement for TonB protein in a variety of membrane-related processes suggests that TonB is an envelope protein. Consistent with this suggestion, the deduced TonB amino acid sequence (Postle, K., and Good R. F., (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 5235-5239) contains an amino-terminal region similar to leader (signal) sequences of exported proteins, although its charged region falls outside the rules which characterize these sequences (von Heijne, G. (1985) J. Mol. Biol. 184, 99-105). The deduced TonB amino acid sequence contains three potential methionine start codons in the first six codons of the open reading frame. In this report, we show, by Edman degradation of [35S]methionine-labeled protein, that TonB protein synthesized in vitro initiates at the third of these methionine codons. A method for detecting TonB synthesized in vivo has been developed that involves expression of TonB from the λP(L) promoter and pulse labeling with [35S]methionine. TonB synthesized in vivo has a chemical half-life of 10 min at 42°C. It is exported from the cytoplasm, as determined by proteinase K accessibility experiments. It fractionates with spheroplasts under conditions where maltose-binding protein fractionates with the periplasm. It has the same mobility in three different polyacrylamide gel systems as TonB synthesized in vitro. We conclude that the amino terminus of TonB is uncleaved following its export from the cytoplasm and that TonB is a membrane-associated protein. Characterization of a tonB-phoA gene fusion suggests that the amino-terminal 41 amino acids of TonB are sufficient to promote export of the fusion protein and presumably TonB as well. Models for TonB orientation within the cell envelope are presented.",
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Escherichia coli TonB protein is exported from the cytoplasm without proteolytic cleavage of its amino terminus. / Postle, Kathleen; Skare, J. T.

In: Journal of Biological Chemistry, Vol. 263, No. 22, 1988, p. 11000-11007.

Research output: Contribution to journalArticle

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