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

Authors

  • William D. Paiva,

    1. Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, Oklahoma 73104, USA.
    2. Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019, USA.
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  • Philip M. Silverman

    Corresponding author
    1. Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, Oklahoma 73104, USA.
    2. Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019, USA.
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Summary

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 (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-amino-acid 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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