The twin arginine translocation pathway in Escherichia coli : mechanism and quality control
Ren, Chao, Ph.D. (2011) The twin arginine translocation pathway in Escherichia coli : mechanism and quality control. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b2565519~S1
The bacterial twin-arginine translocation (Tat) pathway has the unique ability to
export pre-folded proteins across the cytoplasmic membrane. Its name came from the
almost invariant twin-arginine motif in the signal peptide of Tat substrates.
Escherichia coli, a Gram-negative bacterium, is typically used to understand Tat
function in bacteria. Until now, evidence has shown that TatA, TatB and TatC
comprise the minimally functional unit, moreover, a quality control system exists to
monitor the assembly of cofactors and the correctly folding state of proteins which
avoids the futile export and initiates the degradation of rejected molecules.
The research presented in this thesis sought to gain insight into the quality control
mechanism of the Tat pathway in E. coli, and also study the relationship between
transport and maturation of substrates.
In the first place, a novel Tat substrate, YedY, was used to analyse the nature and
variety of proofreading functions operating in conjunction with the Tat pathway. The
single substitutions in three predicted ligands for the YedY molybdopterin centre led
to complete inhibition of export and variable degradation of mutated YedY forms,
indicating an effective proofreading activity. Circular dichroism spectroscopy and
inductively coupled plasma mass spectrometry of purified proteins demonstrated the
change of secondary structures between YedY and mutated variants, and also
indicated the content of Mo in molybdopterin cofactor within proteins. The data
suggested that the three mutated forms failed to correctly assemble cofactor which
resulted in rejection by the Tat export pathway on the basis of the different changes
of secondary structures. Further analysis shows that none of the known export
chaperones for molybdenum cofactor-containing Tat substrates is required for YedY
biogenesis; export is unaffected in cells lacking DmsD and TorD.
In the second place, maturation of pre-YedY was blocked when an Ala>Leu
substitution was made at the -1 position of the signal peptide, and a membrane-bound
precursor form accumulated in the membrane. However, the mature domain had been
transferred to the periplasm. The accumulation did not block transport of other Tat
substrates, indicating the precursor exited from the translocation channel and
integrated into the membrane bilayer. Since the precursor was not detected in the
periplasm, it was suggested that the precursor has undergone lateral transfer into the
bilayer during translocation.
These results are discussed in relation to the overall mechanism of translocation and
proofreading by the Tat pathway in E. coli.
|Item Type:||Thesis or Dissertation (PhD)|
|Subjects:||Q Science > QR Microbiology|
|Library of Congress Subject Headings (LCSH):||Proteins -- Biological transport, Escherichia coli -- Physiology|
|Official Date:||December 2011|
|Institution:||University of Warwick|
|Theses Department:||School of Life Sciences|
|Supervisor(s)/Advisor:||Robinson, Colin, 1958-|
|Extent:||xix, 204 leaves : ill., charts|
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