Part:BBa_K1012002
The TorA twin-arginine signal sequence
DNA encoding the TorA twin-arginine signal peptide from E. coli TMAO reductase. This will target the protein for Tat transport when produced in an E. coli chassis.
Usage and Biology
The E. coli TorA protein is a periplasmic trimethylamine N-oxide (TMAO) reductase (Méjean et al. 1994). The enzyme contains a bis-molybdopterin guanine dinucleotide cofactor (somtimes called MGD or molybdenum cofactor [MoCo]). This enables the enzyme to perform the two-electron reduction of TMAO to trimethylamine (TMA), which has the unmistakable odour of rotting fish. The MGD cofactor is sensitive to oxidation and must be synthesised and inserted into the TorA apoenzyme in the reducing environment of the cell cytoplasm prior to export to the periplasm. Indeed, it was this unusual order of events, which would imply that TorA must be exported in a folded form, that led to the hypothesis that a transport system for folded proteins may exist (Santini et al. 1998). The genes for a transport system dedicated to export of fully folded proteins were described soon after (Sargent et al. 1998).
The key to translocation of the folded TorA protein lies with its unusual N-terminal signal peptide. The TorA signal peptide contains a conserved twin-arginine motif (highlighted in red) that directs the protein to the twin-arginine translocation (Tat) system in the cytoplasmic membrane (reviewed by Palmer & Berks 2012). The TorA Tat signal peptide also has a characteristic AxA motif at its C-terminal end (highlighted in blue) that facilitates cleavage by leader peptidase I (LepB) in E. coli (Palmer & Berks 2012).
1-MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA-39
When attached to other reporter proteins (e.g. GFP, chlormaphenicol acetyl transferase) the TorA signal peptide retains its targeting function and facilitates export of non-native substrates (e.g. Santini et al. 2001).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
The TorA signal peptide can direct export of the human PP1 protein in E. coli.
PP1 encoded by BBa_K1012001 was genetically fused to the E. coli TorA twin-arginine signal peptide encoded by BBa_K1012002 to give a TorASP-PP1 fusion protein. The E. coli K-12 chassis MC4100 was transformed with a plasmid encoding the TorASP-PP1 fusion protein and the cells were fractionated by permeabilisation of the outer membrane and digestion of the cell wall. This generated a periplasmic fraction and a sphaeroplast fraction (which is the cytoplasm of E. coli bounded by an intact inner membrane). The periplasm and sphaeroplast samples were analysed by Western immunoblotting using an HA antibody. As shown in Figure 1A, PP1 was successfully produced when fused to the TorA signal peptide.
Figure 1: PP1 can be exported to the periplasm by the TorA twin-arginine signal peptide. (A) E. coli MC4100 cells producing PP1 (BBa_K1012001) fused to the signal peptides of TorA (TorASP-PP1; Tat-targeting) or MalE (MalESP-PP1; Sec-targeting)* or (B) E. coli strain MC4100 delta-tatABC (which lacks a functional Tat pathway) producing TorASP-PP1, were cultured in LB medium, cells harvested, washed and the periplasmic and sphaeroplast fractions prepared. Samples were separated by SDS-PAGE (15% w/v acrylamide), transferred to PVDF membrane and probed with anti-HA antibody. Samples represent the same proportion of total protein present in the two fractions.
Analysis of the periplasmic fractions from cells producing TorASP-PP1 clearly show the presence of PP1 in the periplasmic fraction when it was supplied with a Tat targeting signal peptide (Figure 1A). To confirm that the Tat pathway was definitely responsible for the transport of PP1 fused to the TorA signal peptide, a mutant of MC4100 lacking a functional Tat pathway (delta-tatABC) was used. The tat mutant E. coli producing the TorASP-PP1 construct was fractionated and analysed by Western immunoblotting. In this case PP1 was present in whole cell and sphaeroplast fractions, but absent in the periplasmic fraction (Figure 1B). Taken together, these data demonstrate that PP1 can transported across the inner membrane by the Tat pathway.
*Note that the data shown here that focus on the use of the MalE signal peptide are related to BBa_K1012004
References
Méjean V, Iobbi-Nivol C, Lepelletier M, Giordano G, Chippaux M, Pascal MC. (1994) TMAO anaerobic respiration in Escherichia coli: involvement of the tor operon. Molecular Microbiology 11:1169-1179.
Santini CL, Ize B, Chanal A, Müller M, Giordano G, Wu LF. (1998) A novel Sec-independent periplasmic protein translocation pathway in Escherichia coli. The EMBO Journal 17:101-112.
Santini CL, Bernadac A, Zhang M, Chanal A, Ize B, Blanco C, Wu LF. (2001) Translocation of jellyfish green fluorescent protein via the Tat system of Escherichia coli and change of its periplasmic localization in response to osmotic up-shock. Journal of Biological Chemistry 276:8159-8164.
Sargent F, Bogsch EG, Stanley NR, Wexler M, Robinson C, Berks BC, Palmer T. (1998) Overlapping functions of components of a bacterial Sec-independent protein export pathway. The EMBO Journal 17:3640-3650.
Palmer T, Berks BC. (2012) The twin-arginine translocation (Tat) protein export pathway. Nature Reviews Microbiology 10:483-496.
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