Structure-function studies of the Tat protein translocation channel
Lead Research Organisation:
Birkbeck, University of London
Department Name: Biological Sciences
Abstract
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Technical Summary
The membrane proteins TatA, TatB and TatC are the essential components of the Tat transport pathway. TatA and TatBC form separate, highly oligomeric, complexes. The TatBC complex functions as a receptor for the substrate protein. TatA is proposed to form the transmembrane protein translocation channel. This suggestion is supported by our recent low resolution structure of the TatA complex, obtained by negative stain electron microscopy, which shows a variable-diameter ring occluded at one end by a lid structure. This project aims to: (a) continue our studies of the structures of the Tat complexes by electron microscopy. Obtain structures for the TatBC complex by particle electron microscopy methods. Correlate structural features of the TatA complex with protein domains by truncation analysis. Produce higher resolution structures of TatA using single particle cryo-electron microscopy. (b) Carry out a multidisciplinary programme of research to test, exploit and expand the structural and mechanistic insights arising from our low resolution structure of the TatA complex. We will: Test models of channel operation by assessing the oligomeric state of native TatA molecules and examining whether TatA complexes exhibit transport-dependent exchange of TatA protomers. Assess using electron microscopy, atomic force microscopy and electrophysiological techniques whether the TatA complex contains an aqueous channel in the native membrane environment. Attempt to identify regions of the TatA protein that must move to allow Tat transport to occur. To do this we will analyse the effects of introduced disulfide bonds (identified in other work) on in vitro protein transport. Joint with BB/C516144/1
People |
ORCID iD |
| Helen Saibil (Principal Investigator) |
Publications
Fronzes R
(2009)
Structure of a type IV secretion system core complex.
in Science (New York, N.Y.)
Krojer T
(2008)
Structural basis for the regulated protease and chaperone function of DegP.
in Nature
Tarry MJ
(2009)
Structural analysis of substrate binding by the TatBC component of the twin-arginine protein transport system.
in Proceedings of the National Academy of Sciences of the United States of America
| Description | The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. In Escherichia coli substrate proteins initially bind to the integral membrane TatBC complex which then recruits the protein TatA to effect translocation. Overproduction of TatBC and the substrate protein SufI in the absence of TatA led to the accumulation of TatBC-SufI complexes that could be purified using an affinity tag on the substrate. Three-dimensional structures of the TatBC-SufI complexes and unliganded TatBC were obtained by single-particle electron microscopy and random conical tilt reconstruction. Comparison of the structures shows that substrate molecules bind on the periphery of the TatBC complex and that substrate binding causes a significant reduction in diameter of the TatBC part of the complex. Although the TatBC complex contains multiple copies of the signal peptide-binding TatC protomer, purified TatBC-SufI complexes contain only 1 or 2 SufI molecules. Where 2 substrates are present in the TatBC-SufI complex, they are bound at adjacent sites. These observations imply that only certain TatC protomers within the complex interact with substrate or that there is a negative cooperativity of substrate binding. Similar TatBC substrate complexes can be generated by an alternative in vitro reconstitution method and using a different substrate protein. |
| Exploitation Route | It is relevant to bacterial and plant physiology and could therefore be applicable to anti bacterials or agriculture. |
| Sectors | Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology |