The majority of outer membrane, periplasmic and some inner membrane proteins of Escherichia coli are synthesised with signal sequences which initiate the translocation process. It has been suggested that other polypeptide sequences within the mature protein carry additional information which determines the final localisation of the product. The aim of this project was to investigate the assembly into the outer membrane of the E. coli ferrichrome receptor protein, TonA. The tonA gene was subcloned onto pBR325 in order to maximise expression of this normally minor outer membrane protein. A study of the kinetics of assembly of TonA in a strain harbouring a multicopy plasmid carrying tonA revealed the occurrence of a processed assembly intermediate which separated with the soluble (cytoplasmic plus periplasmic) fraction of sonicated cells. The position and direction of transcription of tonA was deduced by Tn1000 mutagenesis followed by analysis of the resultant truncated TonA' polypeptides synthesised in vitro and in maxicells. All the TonA' polypeptides thus produced, even those with apparently small C-terminal deletions, fractionated with the sarkosyl soluble envelope material in maxicells (wild type TonA is sarkosyl insoluble), suggesting an important role for the C-terminus in assembly. A similar result was obtained when the tonA gene was truncated using an "oligo-stop translation" sequence. This eliminated the possibility that complete assembly of the TonA' polypeptides truncated by Tn1000 insertion was prevented by Tn1000 encoded sequences at their C-termini. Synthesis of the hybrid MalE-LacZ protein, 72-47, was demonstrated to inhibit the processing of TonA and several inner membrane proteins. Since this hybrid was already known to block the assembly and processing of periplasmic and outer membrane proteins, this result suggests that all three classes of exported protein share common steps in their assembly.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:349721 |
| Date | January 1984 |
| Creators | Jackson, Maria Elizabeth |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/34429 |
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