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Studies on the role of SecA, FtsY and Tfh in the insertion of membrane proteins in Escherichia coli

Henrichs, Tanja 2004. Studies on the role of SecA, FtsY and Tfh in the insertion of membrane proteins in Escherichia coli. PhD Thesis, Cardiff University.

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The essential protein SecA plays a key role in post-translational protein secretion across the inner membrane of Escherichia coli. However, the extent of SecA-requirement in co-translational insertion of inner membrane proteins mediated by the signal recognition particle and its membrane-associated receptor FtsY is not clear. To inactivate SecA function a seven amino acid sequence that is specifically recognised by tobacco etch virus protease was introduced into the secA sequence. The resulting secA-constructs were integrated into the chromosome at the 1 attachment site via a specific lambda phage. Chromosomal wild-type secA was then eliminated by using the 1 Red recombinase system. Cleavage of nascent SecA 195 by a trigger factor-TEV protease hybrid protein resulted in effective inhibition of SecA activity. In addition, SecA830 under the control of the lacpromoter could be depleted in cells not supplemented with the inducer IPTG. Biotinylation assays showed that inactivation of SecA via site-specific proteolysis or depletion of SecA affected the proper secretion of the periplasmic protein alkaline phosphatase as well as the proper biogenesis of the inner membrane proteins FtsQ and MalF274. Similar experiments with Ffh- and FtsY-depletion strains confirmed the involvement of these proteins in inner membrane protein assembly. DNA macroarray technique was used to compare gene expression profiles of cells depleted either of Ffh, FtsY or SecA with the profiles obtained from undepleted cells. While depletion of Ffh and FtsY resulted in the induction of heat shock genes, depletion of SecA induced the expression of the phage-shock protein operon. The results strengthen the notion that SecA not only plays the central role in post-translational secretion of preproteins, but is also extensively involved in co-translational protein translocation. In addition, DNA array analysis indicated a possible involvement of cellular chaperones in targeting of inner membrane proteins.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Biosciences
Subjects: Q Science > QH Natural history > QH301 Biology
Date of First Compliant Deposit: 30 March 2016
Last Modified: 10 Jan 2018 02:46

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