Effects of F-encoded components and F-pilin domains on the synthesis and membrane insertion of TraA′-′PhoA fusion proteins

William Paiva, Philip M. Silverman

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

F-pilin, the 70-amino-acid F-pilus subunit, accumulates in the cell envelope of F + strains in a process that requires interactions between its precursor (the traA gene product) and other host and F-encoded proteins. Here, we have used a set of Φ(traA-phoA) genes to explore the effects of different TraA domains on the synthesis and membrane insertion of TraA-PhoA fusion proteins, particularly in relation to other F-encoded gene products. The 51-amino-acid TraA leader peptide fused directly to alkaline phosphatase was synthesized at comparable rates and incorporated rapidly and efficiently into the inner membrane in F′ and F - cells. A second fusion gene encoded the TraA leader peptide and the first 51 amino acids of F-pilin itself fused to PhoA (TraA′-′PhoA-102 polypeptide). Alkaline phosphatase activities and patterns of pulse-labelled polypeptides indicated that TraA′-′PhoA-102 was synthesized at comparable rates in F′ and F - cells, but in neither was the TraA′-′PhoA-102 polypeptide efficiently processed as a membrane protein. A third gene encoded the entire 121-aminoacid TraA polypeptide fused to PhoA (TraA-′PhoA-121 polypeptide). About 70% of the pulse-labelled TraA-′PhoA-121 polypeptide was rapidly processed in F′ cells, where it accumulated in the cell envelope as active alkaline phosphatase, whereas in F - cells, <5% of the pulse-labelled polypeptide was processed. Additionally, the apparent rate of TraA-′PhoA-121 polypeptide synthesis was threefold higher in F′ cells. The traQ gene alone could not substitute for F in restoring TraA-′PhoA-121 (or wild-type F-pilin) accumulation.

Original languageEnglish
Pages (from-to)1277-1286
Number of pages10
JournalMolecular Microbiology
Volume19
Issue number6
DOIs
StatePublished - 1 Jan 1996

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Fimbriae Proteins
Peptides
Membranes
Proteins
Alkaline Phosphatase
Genes
Protein Sorting Signals
Amino Acids
Gene Fusion
Membrane Proteins

Cite this

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title = "Effects of F-encoded components and F-pilin domains on the synthesis and membrane insertion of TraA′-′PhoA fusion proteins",
abstract = "F-pilin, the 70-amino-acid F-pilus subunit, accumulates in the cell envelope of F + strains in a process that requires interactions between its precursor (the traA gene product) and other host and F-encoded proteins. Here, we have used a set of Φ(traA-phoA) genes to explore the effects of different TraA domains on the synthesis and membrane insertion of TraA-PhoA fusion proteins, particularly in relation to other F-encoded gene products. The 51-amino-acid TraA leader peptide fused directly to alkaline phosphatase was synthesized at comparable rates and incorporated rapidly and efficiently into the inner membrane in F′ and F - cells. A second fusion gene encoded the TraA leader peptide and the first 51 amino acids of F-pilin itself fused to PhoA (TraA′-′PhoA-102 polypeptide). Alkaline phosphatase activities and patterns of pulse-labelled polypeptides indicated that TraA′-′PhoA-102 was synthesized at comparable rates in F′ and F - cells, but in neither was the TraA′-′PhoA-102 polypeptide efficiently processed as a membrane protein. A third gene encoded the entire 121-aminoacid TraA polypeptide fused to PhoA (TraA-′PhoA-121 polypeptide). About 70{\%} of the pulse-labelled TraA-′PhoA-121 polypeptide was rapidly processed in F′ cells, where it accumulated in the cell envelope as active alkaline phosphatase, whereas in F - cells, <5{\%} of the pulse-labelled polypeptide was processed. Additionally, the apparent rate of TraA-′PhoA-121 polypeptide synthesis was threefold higher in F′ cells. The traQ gene alone could not substitute for F in restoring TraA-′PhoA-121 (or wild-type F-pilin) accumulation.",
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Effects of F-encoded components and F-pilin domains on the synthesis and membrane insertion of TraA′-′PhoA fusion proteins. / Paiva, William; Silverman, Philip M.

In: Molecular Microbiology, Vol. 19, No. 6, 01.01.1996, p. 1277-1286.

Research output: Contribution to journalArticle

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AU - Silverman, Philip M.

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N2 - F-pilin, the 70-amino-acid F-pilus subunit, accumulates in the cell envelope of F + strains in a process that requires interactions between its precursor (the traA gene product) and other host and F-encoded proteins. Here, we have used a set of Φ(traA-phoA) genes to explore the effects of different TraA domains on the synthesis and membrane insertion of TraA-PhoA fusion proteins, particularly in relation to other F-encoded gene products. The 51-amino-acid TraA leader peptide fused directly to alkaline phosphatase was synthesized at comparable rates and incorporated rapidly and efficiently into the inner membrane in F′ and F - cells. A second fusion gene encoded the TraA leader peptide and the first 51 amino acids of F-pilin itself fused to PhoA (TraA′-′PhoA-102 polypeptide). Alkaline phosphatase activities and patterns of pulse-labelled polypeptides indicated that TraA′-′PhoA-102 was synthesized at comparable rates in F′ and F - cells, but in neither was the TraA′-′PhoA-102 polypeptide efficiently processed as a membrane protein. A third gene encoded the entire 121-aminoacid TraA polypeptide fused to PhoA (TraA-′PhoA-121 polypeptide). About 70% of the pulse-labelled TraA-′PhoA-121 polypeptide was rapidly processed in F′ cells, where it accumulated in the cell envelope as active alkaline phosphatase, whereas in F - cells, <5% of the pulse-labelled polypeptide was processed. Additionally, the apparent rate of TraA-′PhoA-121 polypeptide synthesis was threefold higher in F′ cells. The traQ gene alone could not substitute for F in restoring TraA-′PhoA-121 (or wild-type F-pilin) accumulation.

AB - F-pilin, the 70-amino-acid F-pilus subunit, accumulates in the cell envelope of F + strains in a process that requires interactions between its precursor (the traA gene product) and other host and F-encoded proteins. Here, we have used a set of Φ(traA-phoA) genes to explore the effects of different TraA domains on the synthesis and membrane insertion of TraA-PhoA fusion proteins, particularly in relation to other F-encoded gene products. The 51-amino-acid TraA leader peptide fused directly to alkaline phosphatase was synthesized at comparable rates and incorporated rapidly and efficiently into the inner membrane in F′ and F - cells. A second fusion gene encoded the TraA leader peptide and the first 51 amino acids of F-pilin itself fused to PhoA (TraA′-′PhoA-102 polypeptide). Alkaline phosphatase activities and patterns of pulse-labelled polypeptides indicated that TraA′-′PhoA-102 was synthesized at comparable rates in F′ and F - cells, but in neither was the TraA′-′PhoA-102 polypeptide efficiently processed as a membrane protein. A third gene encoded the entire 121-aminoacid TraA polypeptide fused to PhoA (TraA-′PhoA-121 polypeptide). About 70% of the pulse-labelled TraA-′PhoA-121 polypeptide was rapidly processed in F′ cells, where it accumulated in the cell envelope as active alkaline phosphatase, whereas in F - cells, <5% of the pulse-labelled polypeptide was processed. Additionally, the apparent rate of TraA-′PhoA-121 polypeptide synthesis was threefold higher in F′ cells. The traQ gene alone could not substitute for F in restoring TraA-′PhoA-121 (or wild-type F-pilin) accumulation.

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