Investigation into the twin-arginine translocation pathway of halophilic and thermophilic archaea

Abstract

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 translocation.

Date of Award	1 Jul 2009
Original language	English
Awarding Institution	University of Bath
Supervisor	Albert Bolhuis (Supervisor)