Mechanisms of protein translocation in Escherichia coli.
thesisposted on 19.11.2015, 08:53 by Karen Anne. Baker
A wide variety of proteins which are synthesised in the cytoplasm of E. coli are subsequently directed either to non-cytoplasmic compartments or transported to the extracellular medium. Proteins which are exported from the cytoplasm are thought to interact with a complex cellular machinery and a number of mutations affecting this secretion machinery have been isolated. In this study, the export of the outer membrane protein TonA was used as a model system to examine the effect on protein translocation of two temperature sensitive secretion mutants, secA and secY. Initial analysis of the effect of secAts mutations on bulk envelope protein synthesis confirmed the key role of SecA in protein transport, including many proteins assembled into the inner membrane. Analysis of the rate of processing of preTonA, pulse-labelled at the restrictive temperature and chased at the permissive temperature revealed differences between SecA and SecY mutants. In particular these data indicate that SecA and SecY may interact sequentially to promote protein export and that SecA may be required to maintain preTonA in a translocationally competent form prior to interaction with SecY. In order to investigate the nature of a specific "export" signal within a protein to be exported, the possibility of using the novel secretion signal at the C-terminus of E. coli haemolysin to direct chimeric protein into the medium was also investigated. The C-terminal signal was successfully fused to a hybrid protein containing a few residues of ss-galactosidase and the majority of E. coli outer membrane protein OmpF lacking its own NH2-terminal signal sequence. The chimeric protein is specifically translocated across the inner and outer membranes and is released into the medium. Consistent with a transport system which bypasses the periplasm, other studies indicated that haemolysin transport is secA independent but may involve secY. Finally, the localisation of haemolysin and several outer membrane proteins synthesised in spheroplasts was also examined in the hope of gaining some further insight into the route taken by proteins which reach the outer membrane or the external medium.