Proof-reading of folded proteins by the Escherichia coli Tat machinery

Lead Research Organisation: University of Dundee
Department Name: School of Life Sciences


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Technical Summary

The Tat protein transport system has the remarkable ability of being able to move folded substrate proteins across the ionically tight bacterial cytoplasmic membrane. We have identified a number of mutations in the TatB component of the Tat machinery that bypasses the proof-reading capacity of the Tat system, thus allowing the transport of both folded and unfolded substrates. We aim to assess how the proof-reading function of the Tat translocase is achieved. We will use a genetic approach to identify further mutations in TatB and in other Tat components that allow export of unfolded substrate proteins. We will focus on mutations in TatB to understand how proof-reading is achieved. Using an in vitro transport assay system we will ascertain whether this is a property of the TatB protein itself, or whether it is mediated by soluble cytoplasmic factors. Finally we will assess whether TatB acts in concert with the TatC protein to carry out its proof-reading function.


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Description Some bacterial proteins operate on the outside of the cell, for example the toxins produced by bacterial pathogens. Since all proteins are made inside the bacterium, the extracellular proteins must be moved out of the cell across the normally impermeable cell membrane. This task is carried out by
machines termed protein transporters that are located in the cell membrane. One type of transporter moves unfolded proteins, threading them across the membrane like string through the eye of a needle. In contrast, a second type of transporter, which we term the Tat system, moves folded proteins across the membrane.
In the bacterium E. coli, the Tat transporter is a large protein complex made up of 3 types of proteins, TatA, TatB and TatC.

In this work we have:

observed that when we overexpress of tatA we see an ability of the Tat system to export unfolded proteins. This was very unexpected and adds to the growing body of evidence that under some circumstances unfolded proteins can be exported by the Tat machinery. It also suggests that the level of TatA is limiting for export of unfolded proteins.

We purified a number of complexes of the TatBC proteins containing different truncated versions of TatB. This gives us valuable information about the interaction domain between the two proteins.

We have generated a bank of E. coli tatB site-directed point mutations and truncations. We have constructed numerous clones for the overexpression and affinity purification of
TatBC complexes containing truncated variants of TatB. These will be useful to the scientific community.
Exploitation Route While the grant proposal is hypothesis the work produced potentially has applied impact:
Knowledge of the Tat mechanism will underpin efforts to exploit the Tat pathway for biotechnological purposes e.g. secretion to aid downstream processing of protein products of commercial or therapeutic utility.
Knowledge of the Tat mechanism will underpin efforts to use Tat as a pathogen-specific and virulence-determining target for novel antimicrobials.

The College of Life Science has a great deal of experience of engaging with industry regarding commercialisation of discoveries. Research and Innovation Services have an office within the CLS research complex and a member of the team attends each formal meeting held by the division. The RIS team have a large number of industrial contacts and close links to Scottish Enterprise. This will help to maximise the impact of all findings of commercial value.

The RIS team also have strong links with the Dundee University Incubator, a facility aimed at housing and nurturing spin-out companies in the biosciences. Related to the incubator, is a collaboration between the publicly- and privately-funded biotechnology sectors in Dundee called Bio-Dundee which aims to connect local academics and industries to allow collaboration. This is achieved through both formal and informal events.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description No notable impact outside of academia. Science and society activities undertaken by the staff employed on this grant
Description BCB 
Organisation University of Oxford
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Joint grant holder.
Collaborator Contribution Sharing reagents and protocols. Exchange of personnel. Sharing results before publication
Impact Many joint papers and joint grant funding.
Start Year 2006