The Twin arginine translocation pathway translocates fully folded proteins across
cellular membranes and is only utilised by proteins that fold before translocation. It
is a unique process that is found in many bacteria, archaea and also in plant
chloroplasts. Investigation of the bacterial and thylakoidal systems has revealed
much of the substrates and the components involved in their translocation.
Unfortunately, there are still many unanswered questions such as how substrates are
directed to the membrane and the actual mechanism of translocation.
This thesis specifically investigates the Tat pathway of halophilic and thermophilic
archaea. To date, there has been a lack of research into the archaeal Tat pathway and
it is possible that there are unique adaptations because of the extreme environments
that these organisms inhabit. Chapter 3 specifically investigates the thermophiles
Sulfolobus solfataricus and Sulfolobus tokodaii and attempts to purify their Tat
complexes. By doing so it was hoped to learn more about the Tat components and
their interactions. Further experiments were also performed to determine if the two S.
solfataricus Tat operons provide specificity to the Tat substrates that translocate.
Four separate areas of the Tat pathway of halophilic archaea (haloarchaea) were
investigated in Chapters 4-7. Firstly, site-directed mutagenesis was used to analyse
the signal peptides of haloarchaeal Tat substrates in more detail. Consequently, the
resulting data led to the use of bioinformatics to analyse the Haloarchaeal signal
peptide. The bioenergetics of the Tat system was then determined by analysing the
effect of a variety of ionophores on translocation of the Tat substrates AmyH and
SptA. Finally, a series of folding and stability assays were used to increase our
understanding of AmyH, which could provide further information on why this
protein, like many other haloarchaeal proteins, requires the Tat pathway for
|Date of Award||1 Jul 2009|
|Supervisor||Albert Bolhuis (Supervisor)|
- protein translocation