Assembly and mechanism of bacterial twin-arginine translocation systems
Baglieri, J. (2012) Assembly and mechanism of bacterial twin-arginine translocation systems. PhD thesis, University of Warwick.
WRAP_THESIS_Baglieri_2012.pdf - Submitted Version
Download (11Mb) | Preview
Official URL: http://webcat.warwick.ac.uk/record=b2582786~S1
The bacterial twin-arginine translocation (Tat) pathway is able to export pre-folded cofactor containing proteins across the cytoplasmic membrane. Tat substrates bear cleavable N-terminal signal peptides that are characterized by the presence of a critical and highly conserved twin-arginine motif which lends the Tat pathway its name. In Escherichia coli and many other Gram-negative bacteria, three integral membrane proteins: TatA, TatB and TatC are essential for Tat-dependent translocation. In contrast Bacillus subtilis possesses a simpler TatAC system which lacks the TatB component. In E. coli the TatA protein assembles into homo-oligomeric complexes that vary considerably in size. The TatA proteins found in B. subtilis do not exhibit the same degree of heterogeneity and this suggested mechanistic differences between the Tat pathways of Gram-negative and Gram-positive bacteria. How the Tat system works is still poorly understood, and the work presented in this thesis sought to gain insights into the assembly and mechanism of E. coli and B. subtilis Tat pathways. This work focused on the study of two previously uncharacterized components: the E. coli TatA paralog TatE subunit and B. subtilis TatAc subunit. In this thesis the purification and characterization of E. coli TatE complexes is reported. Using analytical gel filtration chromatography, blue-native gelelectrophoresis (BN-PAGE) and single-particle analysis of purified TatE complexes, it was found that the TatE complexes are more discrete than the highly heterogeneous TatA complexes. This finding, together with the ability of TatE to support the translocation of the 90-kDa TorA protein, suggested alternative translocation models in which single TatE complexes do not contribute the bulk of the translocation channel, similar to the B. subtilis model. In addition, co-purification and BN-PAGE experiments demonstrated for the first time that TatE interacts with TatA to form TatAE mixed complexes in the membrane, and reveals a completely novel form of Tat complex that might be functionally significant. A soluble population of TatE was also identified in E. coli cell extracts, and phase separation experiments using Triton X-114 suggested it may be mis-localized. In a separate set of studies, the ability of the B. subtilis TatAc protein to form active translocases in combination with the B. subtilis TatCd or TatCy proteins was investigated for the first time. The TatAcCd and TatAcCy mixed translocases were able to translocate several E. coli Tat substrates including, TorA, AmiA and AmiC. Finally BN-PAGE and gel filtration chromatography showed that the TatAcCd and TatAcCy complexes were significantly smaller than the previously described E. coli TatABC substrate-binding complex.
|Item Type:||Thesis or Dissertation (PhD)|
|Subjects:||Q Science > QR Microbiology|
|Library of Congress Subject Headings (LCSH):||Translocation (Genetics), Escherichia coli -- Physiology, Bacillus subtilis -- Physiology|
|Institution:||University of Warwick|
|Theses Department:||School of Life Sciences|
|Supervisor(s)/Advisor:||Robinson, Colin, 1958-|
|Sponsors:||Seventh Framework Programme (European Commission) (FP7/2007-2013)|
|Extent:||xvii, 167,  leaves : ill.|
Actions (login required)