The Escherichia coli twin-arginine translocation (Tat) system transports fully folded and assembled proteins across the inner membrane into the periplasmic space. The E. coli Tat machinery minimally consists of three integral membrane proteins: TatA, TatB and TatC. A popular model of Tat translocation is that cargo first interacts with a substrate binding complex composed of TatB and TatC and then is transported across the inner membrane through a channel comprised primarily of TatA.
The most common method for observing the kinetics of Tat transport, a protease protection assay, lacks the ability to distinguish between individual transport sub-steps and is limited by the inability to observe translocation in real-time. Therefore, a real-time FRET based assay was developed to observe interactions between the cargo protein pre-SufI, and its initial binding site, the TatBC complex. The cargo was found to first associate with the TatBC complex, and then, in the presence of a membrane potential (∆psi), migrate away from the initial binding site after a 20-45 second delay. Since cargo migration away from the TatBC complex was not directly promoted by the presence of a ∆psi, the delay likely represents some preparatory step that results in a transport competent translocon.
In addition, the Tat system has long been identified as a potential biotechnological tool for protein production. However, much is still unknown about which cargos are suitable for transport by the Tat system. To probe the Tat system’s ability to transport substrates of different sizes and shapes, 18 different cargos were generated using the natural Tat substrate pre-SufI as a base. Transport efficiencies for these cargos indicate that not only is the Tat machinery’s ability to transport substrates determined by the protein’s molecular weight, as well as by its dimensions.
In total, these results suggest a dynamic translocon that undergoes functionally significant, ∆psi-dependent changes during translocation. Moreover, not every protein cargo can be directed through the Tat translocon by a Tat signal peptide, and this selectivity is not only related to the overall size of the protein, but also dependent on shape.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/148188 |
| Date | 14 March 2013 |
| Creators | Whitaker, Neal William 1982- |
| Contributors | Musser, Siegfried M, Scholtz, John M, Pellois, Jean-Philippe, Benedik, Michael J |
| Source Sets | Texas A and M University |
| Detected Language | English |
| Type | Thesis, text |
| Format | application/pdf |
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