Investigation into the role of chaperones in the secretion of haemolysin from Escherichia coli

• Main Author: Whitehead, J. P.
• Published: University of Liverpool 1994
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.508367

Haemolysin is a 107 kDa protein which is secreted independently of the general export pathway. It contains a C-terminal signal which directs its secretion through a trans-envelope translocator, comprised of the proteins HlyB, HlyD and TolC. It was reasonsed that because haemolysin is exported post-translationally it may interact with molecular chaperones to maintain a 'loosely folded' secretion-competent conformation. Investigations carried out indicate that the general export chaperone SecB is not required for the efficient secretion of haemolysin. Preliminary studies using a secB null mutant, in which secretion was significantly reduced, suggested that SecB was essential for efficient secretion. However, further assays using a SecB sequestering approach and complementation of the secB null mutant indicated that SecB is not required either directly, to modulate haemolysin folding, or indirectly in the assembly of the membrane translocator. The reduced secretion by the secB null mutant is probably due to the pleiotropic effects of the mutation. The SecB sequestering approach has also been reproduced in a T7 expression strain using a newly constructed T7 sequesterer. Following further characterisation, this T7 sequestering approach may be used in pulse-chase experiments without the need for immunoprecipitation, enabling the requirements of exported proteins for SecB to be determined in the absence of specific antisera. Further investigations into the possible requirement of chaperones in the secretion of haemolysin have shown the presence of the fully functional chaperonin GroEL to be a strict requirement for efficient secretion of haemolysin. Using a temperature-sensitive groEL mutant, the level of haemolysin secretion was dramatically reduced at both the permissive and non-permissive temperatures.