Effects of F-encoded components and F-pilin domains on the synthesis and membrane insertion of TraA'-'PhoA fusion proteins.
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 phi(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.
Paiva
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WD
Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City 73104, USA.
Silverman
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PM
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D036781:Fimbriae Proteins; D005798:Genes, Bacterial; D015394:Molecular Structure; D018933:Pili, Sex / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C444040:F pilin, E coli; D011993:Recombinant Fusion Proteins; D036781:Fimbriae Proteins