Guerrero‐Montero, I., Richards, K., Jawara, C., Browning, D., Peswani, A., Labrit, M., Allen, M., Aubry, C., Davé, E., Humphreys, D., Busby, S. and Robinson, C. (2019). Escherichia coli ‘TatExpress’ strains export several g/L human growth hormone to the periplasm by the Tat pathway. Biotechnology and Bioengineering [Online]. Available at: https://doi.org/10.1002/bit.27147.
Escherichia coli is a heavily used platform for the production of biotherapeutic and other high‐value proteins, and a favoured strategy is to export the protein of interest to the periplasm in order to simplify downstream processing and facilitate disulphide bond formation. The Sec pathway is the standard means of transporting the target protein but it is unable to transport complex or rapidly folding proteins because the Sec system can only transport proteins in an unfolded state. The Tat system also operates to transport proteins to the periplasm, and it has significant potential as an alternative means of recombinant protein production because it transports fully folded proteins. Here, we have tested the Tat system's full potential for the production of biotherapeutics for the first time using fed‐batch fermentation. We expressed human growth hormone (hGH) with a Tat signal peptide in E. coli W3110 'TatExpress' strains that contain elevated levels of the Tat apparatus. This construct contained 4 amino acids from TorA at the hGH N‐terminus as well as the initiation methionine from hGH, which is removed in vivo. We show that the protein is efficiently exported to the periplasm during extended fed‐batch fermentation, to the extent that it is by far the most abundant protein in the periplasm. The protein was shown to be homogeneous, disulphide bonded and active. The bioassay showed that the yields of purified periplasmic hGH are 5.4 g/L culture whereas an ELISA gave a figure of 2.39 g/L. Separate analysis of a TorA‐hGH construct lacking any additional amino acids likewise showed efficient export to the periplasm, although yields were approximately 2‐fold lower.
Frain, K., Robinson, C. and van Dijl, J. (2019). Transport of Folded Proteins by the Tat System. The Protein Journal [Online]. Available at: https://doi.org/10.1007/s10930-019-09859-y.
The twin-arginine protein translocation (Tat) system has been characterized in bacteria, archaea and the chloroplast thylakoidal membrane. This system is distinct from other protein transport systems with respect to two key features. Firstly, it accepts cargo proteins with an N-terminal signal peptide that carries the canonical twin-arginine motif, which is essential for transport. Second, the Tat system only accepts and translocates fully folded cargo proteins across the respective membrane. Here, we review the core essential features of folded protein transport via the bacterial Tat system, using the three-component TatABC system of Escherichia coli and the two-component TatAC systems of Bacillus subtilis as the main examples. In particular, we address features of twin-arginine signal peptides, the essential Tat components and how they assemble into different complexes, mechanistic features and energetics of Tat-dependent protein translocation, cytoplasmic chaperoning of Tat cargo proteins, and the remarkable proofreading capabilities of the Tat system. In doing so, we present the current state of our understanding of Tat-dependent protein translocation across biological membranes, which may serve as a lead for future investigations.
Frain, K., van Dijl, J. and Robinson, C. (2019). The Twin-Arginine Pathway for Protein Secretion. EcoSal Plus [Online] 8. Available at: https://doi.org/10.1128/ecosalplus.ESP-0040-2018.
The Tat pathway for protein translocation across bacterial membranes stands out for its selective
handling of fully folded cargo proteins. In this review, we provide a comprehensive summary of our
current understanding of the different known Tat components, their assembly into different
complexes and their specific roles in the protein translocation process. In particular, this overview
focuses on the Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Bacillus
subtilis. Using these organisms, we discuss structural features of Tat complexes alongside
mechanistic models that allow for the Tat pathway’s unique protein proofreading and transport
capabilities. Finally, we highlight recent advances in exploiting the Tat pathway for biotechnological
benefit, the production of high value pharmaceutical proteins.
Browning, D., Godfrey, R., Richards, K., Robinson, C. and Busby, S. (2019). Exploitation of the Escherichia coli lac operon promoter for controlled recombinant protein production. Biochemical Society Transactions [Online] 47:755-763. Available at: https://doi.org/10.1042/BST20190059.
The Escherichia coli lac operon promoter is widely used as a tool to control recombinant protein production in bacteria. Here we give a brief review of how it functions, how it is regulated, and how, based on this knowledge, a suite of lac promoter derivatives has been developed to give controlled expression that is suitable for diverse biotechnology applications.
Charoonnart, P., Worakajit, N., Zedler, J., Meetam, M., Robinson, C. and Saksmerprome, V. (2019). Generation of microalga Chlamydomonas reinhardtii expressing shrimp antiviral dsRNA without supplementation of antibiotics. Scientific Reports [Online] 9:3164. Available at: https://doi.org/10.1038/s41598-019-39539-x.
RNA interference (RNAi) is an efective way of combating shrimp viruses by using sequence-specifc
double-stranded (dsRNA) designed to knock down key viral genes. The aim of this study was to use
microalgae expressing antiviral dsRNA as a sustainable feed supplement for shrimp ofering viral
protection. In this proof of concept, we engineered the chloroplast genome of the green microalga
Chlamydomonas reinhardtii for the expression of a dsRNA cassette targeting a shrimp yellow head
viral gene. We used a previously described chloroplast transformation approach that allows for
the generation of stable, marker-free C. reinhardtii transformants without the supplementation of
antibiotics. The generated dsRNA-expressing microalgal strain was then used in a shrimp feeding
trial to evaluate the efciency of the algal RNAi-based vaccine against the virus. Shrimps treated
with dsRNA-expressed algal cells prior to YHV infection had 50% survival at 8 day-post infection
(dpi), whereas 84.1% mortality was observed in control groups exposed to the YHV virus. RT-PCR
using viral specifc primers revealed a lower infection rate in dsRNA-expressing algae treated shrimp
(55.6±11.1%) compared to control groups (88.9±11.1% and 100.0±0.0%, respectively). Our
results are promising for using microalgae as a novel, sustainable alternative as a nutritious, anti-viral
protective feedstock in shrimp aquaculture.
Guerrero Montero, I., Magdalena Dolata, K., Schlüter, R., Malherbe, G., Sievers, S., Zühlke, D., Sura, T., Dave, E., Riedel, K. and Robinson, C. (2019). Comparative proteome analysis in an Escherichia coli CyDisCo strain identifies stress responses related to protein production, oxidative stress and accumulation of misfolded protein. Microbial Cell Factories [Online] 18. Available at: http://dx.doi.org/10.1186/s12934-019-1071-7.
The Twin-arginine translocation (Tat) pathway of Escherichia coli has great potential for the export of biopharmaceuticals to the periplasm due to its ability to transport folded proteins, and its proofreading mechanism that allows correctly folded proteins to translocate. Coupling the Tat-dependent protein secretion with the formation of disulfide bonds in the cytoplasm of E. coli CyDisCo provides a powerful platform for the production of industrially challenging proteins. In this study, we investigated the effects on the E. coli cells of exporting a folded substrate (scFv) to the periplasm using a Tat signal peptide, and the effects of expressing an export-incompetent midsfolded variant.
Cell growth is decreased when either the correctly folded or misfolded scFv is expressed with a Tat signal peptide. However, only the production of misfolded scFv leads to cell aggregation and formation of inclusion bodies. The comprehensive proteomic analysis revealed that both conditions, recombinant protein overexpression and misfolded protein accumulation, lead to downregulation of membrane transporters responsible for protein folding and insertion into the membrane while upregulating the production of chaperones and proteases involved in removing aggregates. These conditions also differentially affect the production of transcription factors and proteins involved in DNA replication. The most distinct stress response observed was the cell aggregation caused by elevated levels of antigen 43. Finally, Tat-dependent secretion causes an increase in tatA expression only after induction of protein expression, while the subsequent post-induction analysis revealed lower tatA and tatB expression levels, which correlate with lowered TatA and TatB protein abundance.
The study identified characteristic changes occurring as a result of the production of both a folded and a misfolded protein, but also highlights an exclusive unfolded stress response. Countering and compensating for these changes may result in higher yields of pharmaceutically relevant proteins exported to the periplasm.
Dolata, K., Montero, I., Miller, W., Sievers, S., Sura, T., Wolff, C., Schlüter, R., Riedel, K. and Robinson, C. (2019). Far-reaching cellular consequences of tat deletion in Escherichia coli revealed by comprehensive proteome analyses. Microbiological Research [Online] 218:97-107. Available at: https://doi.org/10.1016/j.micres.2018.10.008.
In Escherichia coli, the Twin-arginine translocation (Tat) pathway secretes a set of folded proteins
with important physiological functions to the periplasm and outer membrane. The loss of Tat
secretion impairs outer membrane integrity and leads to decreased cell growth. Only recently, the
Tat pathway has gained more attention due to its essential role in bacterial virulence and
applications in the production of fully folded heterologous proteins. In this study, we investigated
the influence of the deletion of all active Tat pathway components on the E. coli cells. The
comprehensive proteomic analysis revealed activation of several stress responses and
experimentally confirmed the dependence of certain proteins on the Tat system for export. We
observed that a tat deletion triggers protein aggregation, membrane vesiculation, synthesis of
colanic acid and biofilm formation. Furthermore, the mislocalization of Tat-dependent proteins
disturbs iron and molybdenum homeostasis and impairs the cell envelope integrity. The results
show that the functional Tat pathway is important for the physiological stability and that its
dysfunction leads to a series of severe changes in E. coli cells.
Sutherland, G., Grayson, K., Adams, N., Mermans, D., Jones, A., Robertson, A., Auman, D., Brindley, A., Sterpone, F., Tuffery, P., Derreumaux, P., Dutton, P., Robinson, C., Hitchcock, A. and Hunter, C. (2018). Probing the quality control mechanism of theEscherichia colitwin-arginine translocase with folding variants of ade novo-designed heme protein. Journal of Biological Chemistry [Online] 293:6672-6681. Available at: https://doi.org/10.1074/jbc.RA117.000880.
Protein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin arginine translocase (Tat). The Tat machinery exports folded and cofactor containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the model Escherichia coli Tat system to recognize and translocate de novo-designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one or no heme b cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional 1H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the E. coli trimethylamine-N-oxide (TMAO) reductase (TorA) to the N-terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of heme b-induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system’s quality control mechanism.
Lima, S., Webb, C., Deery, E., Robinson, C. and Zedler, J. (2018). Human Intrinsic Factor Expression for Bioavailable Vitamin B12 Enrichment in Microalgae. Biology [Online] 7:19. Available at: https://doi.org/10.3390/biology7010019.
Dietary supplements and functional foods are becoming increasingly popular complements to regular diets. A recurring ingredient is the essential cofactor vitamin B12 (B12). Microalgae are making their way into the dietary supplement and functional food market but do not produce B12, and their B12 content is very variable. In this study, the suitability of using the human B12-binding protein intrinsic factor (IF) to enrich bioavailable B12 using microalgae was tested. The IF protein was successfully expressed from the nuclear genome of the model microalga Chlamydomonas reinhardtii and the addition of an N-terminal ARS2 signal peptide resulted in efficient IF secretion to the medium. Co-abundance of B12 and the secreted IF suggests the algal produced IF protein is functional and B12-binding. Utilizing IF expression could be an efficient tool to generate B12-enriched microalgae in a controlled manner that is suitable for vegetarians and, potentially, more bioavailable for humans.
Smith, S., Walker, K., Jones, A., Smith, C. and Robinson, C. (2018). Characterisation of a novel method for the production of single?span membrane proteins in Escherichia coli. Biotechnology and Bioengineering [Online]. Available at: https://doi.org/10.1002/bit.26895.
The large?scale production and isolation of recombinant protein is a central element of the biotechnology industry and many of the products have proved extremely beneficial for therapeutic medicine. Escherichia coli is the microorganism of choice for the expression of heterologous proteins for therapeutic application, and a range of high?value proteins have been targeted to the periplasm using the well characterised Sec protein export pathway. More recently, the ability of the second mainstream protein export system, the twin?arginine translocase, to transport fully?folded proteins into the periplasm of not only E. coli, but other Gram?negative bacteria, has captured the interest of the biotechnology industry.
In this study, we have used a novel approach to block the export of a heterologous Tat substrate in the later stages of the export process, and thereby generate a single?span membrane protein with the soluble domain positioned on the periplasmic side of the inner membrane. Biochemical and immuno?electron microscopy approaches were used to investigate the export of human growth hormone by the twin?arginine translocase, and the generation of a single span membrane?embedded variant. This is the first time that a bona?fide biotechnologically?relevant protein has been exported by this machinery and visualised directly in this manner. The data presented here demonstrate a novel method for the production of single?span membrane proteins in E. coli.
Browning, D., Richards, K., Peswani, A., Roobol, J., Busby, S. and Robinson, C. (2017). Escherichia coli ‘TatExpress’ strains super-secrete human growth hormone into the bacterial periplasm by the Tat pathway. Biotechnology and Bioengineering [Online] 114:2828-2836. Available at: http://dx.doi.org/10.1002/bit.26434.
Numerous high-value proteins are secreted into the Escherichia coli periplasm by the General Secretory (Sec) pathway, but Sec-based production chassis cannot handle many potential target proteins. The Tat pathway offers a promising alternative because it transports fully folded proteins; however, yields have been too low for commercial use. To facilitate Tat export, we have engineered the TatExpress series of super-secreting strains by introducing the strong inducible bacterial promoter, ptac, upstream of the chromosomal tatABCD operon, to drive its expression in E. coli strains commonly used by industry (e.g. W3110 and BL21). This modification significantly improves the Tat-dependent secretion of human growth hormone (hGH) into the bacterial periplasm, to the extent that secreted hGH is the dominant periplasmic protein after only 1?h induction. TatExpress strains accumulate in excess of 30?mg?L?1 periplasmic recombinant hGH, even in shake flask cultures. A second target protein, an scFv, is also shown to be exported at much higher rates in TatExpress strains
Henriques de Jesus, M., Zygadlo Nielsen, A., Busck Mellor, S., Matthes, A., Burow, M., Robinson, C. and Erik Jensen, P. (2017). Tat proteins as novel thylakoid membrane anchors organize a biosynthetic pathway in chloroplasts and increase product yield 4-fold. Metabolic Engineering [Online] 44:108-116. Available at: https://doi.org/10.1016/j.ymben.2017.09.014.
Photosynthesis drives the production of ATP and NADPH, and acts as a source of carbon for primary metabolism. NADPH is also used in the production of many natural bioactive compounds. These are usually synthesized in low quantities and are often difficult to produce by chemical synthesis due to their complex structures. Some of the crucial enzymes catalyzing their biosynthesis are the cytochromes P450 (P450s) situated in the endoplasmic reticulum (ER), powered by electron transfers from NADPH. Dhurrin is a cyanogenic glucoside and its biosynthesis involves a dynamic metabolon formed by two P450s, a UDP-glucosyltransferase (UGT) and a P450 oxidoreductase (POR). Its biosynthetic pathway has been relocated to the chloroplast where ferredoxin, reduced through the photosynthetic electron transport chain, serves as an efficient electron donor to the P450s, bypassing the involvement of POR. Nevertheless, translocation of the pathway from the ER to the chloroplast creates other difficulties, such as the loss of metabolon formation and intermediate diversion into other metabolic pathways. We show here that co-localization of these enzymes in the thylakoid membrane leads to a significant increase in product formation, with a concomitant decrease in off-pathway intermediates. This was achieved by exchanging the membrane anchors of the dhurrin pathway enzymes to components of the Twin-arginine translocation pathway, TatB and TatC, which have self-assembly properties. Consequently, we show 4-fold increased titers of dhurrin and a decrease in the amounts of intermediates and side products in Nicotiana benthamiana. Further, results suggest that targeting the UGT to the membrane is a key factor to achieve efficient substrate channeling.
Velez-Suberbie, M., Betts, J., Walker, K., Robinson, C., Zoro, B. and Keshavarz-Moore, E. (2017). High throughput automated microbial bioreactor system used for clone selection and rapid scale-down process optimization. Biotechnology Progress [Online]. Available at: http://dx.doi.org/10.1002/btpr.2534.
High throughput automated fermentation systems have become a useful tool in early bioprocess development. In this study, we investigated a 24 x 15 mL single use microbioreactor system, ambr 15f, designed for microbial culture. We compared the fed-batch growth and production capabilities of this system for two Escherichia coli strains, BL21 (DE3) and MC4100, and two industrially relevant molecules, hGH and scFv. In addition, different carbon sources were tested using bolus, linear or exponential feeding strategies, showing the capacity of the ambr 15f system to handle automated feeding. We used power per unit volume (P/V) as a scale criterion to compare the ambr 15f with 1 L stirred bioreactors which were previously scaled-up to 20 L with a different biological system, thus showing a potential 1,300 fold scale comparability in terms of both growth and product yield. By exposing the cells grown in the ambr 15f system to a level of shear expected in an industrial centrifuge, we determined that the cells are as robust as those from a bench scale bioreactor. These results provide evidence that the ambr 15f system is an efficient high throughput microbial system that can be used for strain and molecule selection as well as rapid scale-up
Smith, S., Yarwood, A., Fleck, R., Robinson, C. and Smith, C. (2017). TatA complexes exhibit a marked change in organisation in response to expression of the TatBC complex. Biochemical Journal [Online] 474:1495-1508. Available at: http://dx.doi.org/10.1042/BCJ20160952.
The twin arginine translocation (Tat) system is an integral membrane protein complex that accomplishes the remarkable feat of transporting large, fully-folded polypeptides across the inner membrane of bacteria, into the periplasm. In Escherichia coli Tat is comprised of three membrane proteins: TatA, TatB and TatC. How these proteins arrange themselves in the inner membrane to permit passage of Tat substrates, whilst maintaining membrane integrity, is still poorly understood. TatA is the most abundant component of this complex and facilitates assembly of the transport mechanism. We have utilised immunogold labelling in combination with array tomography to gain insight into the localisation and distribution of the TatA protein in E. coli cells. We show that TatA exhibits a uniform distribution throughout the inner membrane of E. coli and that altering the expression of TatBC shows a previously uncharacterised distribution of TatA in the inner membrane. Array tomography was used to provide our first insight into this altered distribution of TatA in 3D space, revealing that this protein forms linear clusters in the inner membrane of E. coli upon increased expression of TatBC. This is the first indication that TatA organisation in the inner membrane alters in response to changes in Tat subunit stoichiometry.
Frain, K., Jones, A., Schoner, R., Walker, K. and Robinson, C. (2016). The Bacillus subtilis TatAdCd system exhibits an extreme level of substrate selectivity. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research [Online] 1864:202-208. Available at: http://dx.doi.org/10.1016/j.bbamcr.2016.10.018.
The Tat system preferentially transports correctly folded proteins across the bacterial membrane although little is known of the proofreading mechanism. Most research has focused on TatABC systems from Gram-negative bacteria, especially Escherichia coli, and much less is known of the TatAC-type systems from Gram-positive organisms. We have previously shown that the Bacillus subtilis TatAdCd system is functional in an E. coli tat null background and able to transport TorA-GFP and native TorA (TMAO reductase); here, we examined its ability to transport other proteins bearing a TorA signal sequence. We show that whereas E. coli TatABC transports a wide range of biotherapeutics including human growth hormone, interferon ?2b, a VH domain protein and 2 different scFvs, TatAdCd transports the scFvs but completely rejects the other proteins. The system also rejects two native E. coli substrates, NrfC and FhuD. Moreover, we have shown that TatABC will transport a wide range of folded scFv variants with the surface altered to incorporate multiple salt bridges, charged residues (5 glutamate, lysine or arginine), or hydrophobic residues (up to 6 leucines). In contrast, TatAdCd completely rejects many of these variants including those with 5 or 6 added Leu residues. The combined data show that the TatABC and TatAdCd systems have very different substrate selectivities, with the TatAdCd system displaying an extreme level of selectivity when compared to the E. coli system. The data also provide a preliminary suggestion that TatAdCd may not tolerate surface domains with a level of hydrophobicity above a certain threshold.
Jones, A., Austerberry, J., Dajani, R., Warwicker, J., Curtis, R., Derrick, J. and Robinson, C. (2016). Proofreading of substrate structure by the Twin-Arginine Translocase is highly dependent on substrate conformational flexibility but surprisingly tolerant of surface charge and hydrophobicity changes. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research [Online] 1863:3116-3124. Available at: http://doi.org/10.1016/j.bbamcr.2016.09.006.
The Tat system transports folded proteins across the bacterial plasma membrane, and in Escherichia coli preferentially transports correctly-folded proteins. Little is known of the mechanism by which Tat proofreads a substrate's conformational state, and in this study we have addressed this question using a heterologous single-chain variable fragment (scFv) with a defined structure. We introduced mutations to surface residues while leaving the folded structure intact, and also tested the importance of conformational flexibility. We show that while the scFv is stably folded and active in the reduced form, formation of the 2 intra-domain disulphide bonds enhances Tat-dependent export 10-fold, indicating Tat senses the conformational flexibility and preferentially exports the more rigid structure. We further show that a 26-residue unstructured tail at the C-terminus blocks export, suggesting that even this short sequence can be sensed by the proofreading system. In contrast, the Tat system can tolerate significant changes in charge or hydrophobicity on the scFv surface; substitution of uncharged residues by up to 3 Lys-Glu pairs has little effect, as has the introduction of up to 5 Lys or Glu residues in a confined domain, or the introduction of a patch of 4 to 6 Leu residues in a hydrophilic region. We propose that the proofreading system has evolved to sense conformational flexibility and detect even very transiently-exposed internal regions, or the presence of unfolded peptide sections. In contrast, it tolerates major changes in surface charge or hydrophobicity.
Zedler, J., Mullineaux, C. and Robinson, C. (2016). Efficient targeting of recombinant proteins to the thylakoid lumen in Chlamydomonas reinhardtii using a bacterial Tat signal peptide. Algal Research [Online] 19:57-62. Available at: http://doi.org/10.1016/j.algal.2016.07.007.
Interest in the exploitation of microalgae for biotechnological applications has increased over the last decade, and microalgae are now viewed as offering a sustainable alternative to traditionally used host chassis. A number of recombinant proteins have been expressed in genetically modified algal strains, with the green alga Chlamydomonas reinhardtii being a particularly popular host strain. While nuclear transformation is possible with this organism, chloroplast transformation offers more reliable expression, and several proteins have been expressed in the stroma. Here, we present the first utilisation of the thylakoid lumen for recombinant protein production in microalgae. A bacterial export signal peptide was used to efficiently translocate two recombinant proteins, a fluorescent reporter protein (pHRed) and a biopharmaceutical model substrate (scFv) into the thylakoid lumen. This approach expands the algal chloroplast genetic toolkit and offers a means of expressing proteins that are difficult to express in the stroma for reasons of toxicity, stability or a requirement for disulphide bonding.
Zedler, J., Gangl, D., Hamberger, B., Purton, S. and Robinson, C. (2015). Stable expression of a bifunctional diterpene synthase in the chloroplast of Chlamydomonas reinhardtii. Journal of Applied Phycology [Online] 27:2271-2277. Available at: http://doi.org/10.1007/s10811-014-0504-2.
Chlamydomonas reinhardtii has been shown to hold significant promise as a production platform for recombinant proteins, but transformation of the nuclear genome is still a non-trivial process due to random gene insertion and frequent silencing. Insertion of transgenes into the chloroplasts is an alternative strategy, and we report here the stable expression of a large (91 kDa) protein in the chloroplast using a recently developed low-cost transformation protocol. Moreover, selection of transformants is based on restoration of prototrophy using an endogenous gene (psbH) as the marker, thereby allowing the generation of transgenic lines without the use of antibiotic-resistance genes. Here, we have expressed a bifunctional diterpene synthase in C. reinhardtii chloroplasts. Homoplasmic transformants were obtained with the expressed enzyme accounting for 3.7 % of total soluble protein. The enzyme was purified to homogeneity and expression was shown to have a small but reproducible effect on growth rate at the end of log phase growth. These results demonstrate that large recombinant enzymes can be synthesised in the algal chloroplast, and serve to underline its potential as a platform for the biosynthesis of novel metabolites.
Gangl, D., Zedler, J., Rajakumar, P., Martinez, E., Riseley, A., W?odarczyk, A., Purton, S., Sakuragi, Y., Howe, C., Jensen, P. and Robinson, C. (2015). Biotechnological exploitation of microalgae. Journal of Experimental Botany [Online] 66:6975-6990. Available at: http://doi.org/10.1093/jxb/erv426.
Microalgae are a diverse group of single-cell photosynthetic organisms that include cyanobacteria and a wide range of eukaryotic algae. A number of microalgae contain high-value compounds such as oils, colorants, and polysaccharides, which are used by the food additive, oil, and cosmetic industries, among others. They offer the potential for rapid growth under photoautotrophic conditions, and they can grow in a wide range of habitats. More recently, the development of genetic tools means that a number of species can be transformed and hence used as cell factories for the production of high-value chemicals or recombinant proteins. In this article, we review exploitation use of microalgae with a special emphasis on genetic engineering approaches to develop cell factories, and the use of synthetic ecology approaches to maximize productivity. We discuss the success stories in these areas, the hurdles that need to be overcome, and the potential for expanding the industry in general.
Alanen, H., Walker, K., Lourdes Velez Suberbie, M., Matos, C., Bönisch, S., Freedman, R., Keshavarz-Moore, E., Ruddock, L. and Robinson, C. (2015). Efficient export of human growth hormone, interferon alpha2b and antibody fragments to the periplasm by the Escherichia coli Tat pathway in the absence of prior disulfide bond formation. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research [Online] 1853:756-763. Available at: http://dx.doi.org/10.1016/j.bbamcr.2014.12.027.
Gangl, D., Zedler, J., W?odarczyk, A., Jensen, P., Purton, S. and Robinson, C. (2015). Expression and membrane-targeting of an active plant cytochrome P450 in the chloroplast of the green alga Chlamydomonas reinhardtii. Phytochemistry [Online] 110:22-28. Available at: http://dx.doi.org/10.1016/j.phytochem.2014.12.006.
Patel, R., Vasilev, C., Beck, D., Monteferrante, C., van Dijl, J., Hunter, C., Smith, C. and Robinson, C. (2014). A mutation leading to super-assembly of twin-arginine translocase (Tat) protein complexes. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research [Online] 1843:1978-86. Available at: http://dx.doi.org/10.1016/j.bbamcr.2014.05.009.
The Tat system transports folded proteins across the bacterial plasma membrane. The mechanism is believed to involve coalescence of a TatC-containing unit with a separate TatA complex, but the full translocation complex has never been visualised and the assembly process is poorly defined. We report the analysis of the Bacillus subtilis TatAyCy system, which occurs as separate TatAyCy and TatAy complexes at steady state, using single-particle electron microscopy (EM) and advanced atomic force microscopy (AFM) approaches. We show that a P2A mutation in the TatAy subunit leads to apparent super-assembly of Tat complexes. Purification of TatCy-containing complexes leads to a large increase in the TatA:TatC ratio, suggesting that TatAy(P2A) complexes may have attached to the TatAyCy complex. EM and AFM analyses show that the wild-type TatAyCy complex purifies as roughly spherical complexes of 9-16nm diameter, whereas the P2A mutation leads to accumulation of large (up to 500nm long) fibrils that are chains of numerous complexes. Time lapsed AFM imaging, recorded on fibrils under liquid, shows that they adopt a variety of tightly curved conformations, with radii of curvature of 10-12nm comparable to the size of single TatAy(P2A) complexes. The combined data indicate that the mutation leads to super-assembly of TatAy(P2A) complexes and we propose that an individual TatAy(P2A) complex assembles initially with a TatAy(P2A)Cy complex, after which further TatAy(P2A) complexes attach to each other. The data further suggest that the N-terminal extracytoplasmic domain of TatAy plays an essential role in Tat complex interactions.
Patel, R., Smith, S. and Robinson, C. (2014). Protein transport by the bacterial Tat pathway. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research [Online] 1843:1620-1628. Available at: http://dx.doi.org/10.1016/j.bbamcr.2014.02.013.
The twin-arginine translocation (Tat) system accomplishes the remarkable feat of translocating large – even dimeric – proteins across tightly sealed energy-transducing membranes. All of the available evidence indicates that it is unique in terms of both structure and mechanism; however its very nature has hindered efforts to probe the core translocation events. At the heart of the problem is the fact that two large sub-complexes are believed to coalesce to form the active translocon, and ‘capturing’ this translocation event has been too difficult. Nevertheless, studies on the individual components have come a long way in recent years, and structural studies have reached the point where educated guesses can be made concerning the most interesting aspects of Tat. In this article we review these studies and the emerging ideas in this field. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.
Matos, C., Robinson, C., Alanen, H., Prus, P., Uchida, Y., Ruddock, L., Freedman, R. and Keshavarz-Moore, E. (2014). Efficient export of prefolded, disulfide-bonded recombinant proteins to the periplasm by the Tat pathway in Escherichia coli CyDisCo strains. Biotechnology Progress [Online] 30:281-290. Available at: http://dx.doi.org/10.1002/btpr.1858.
Albiniak, A., Matos, C., Branston, S., Freedman, R., Keshavarz-Moore, E. and Robinson, C. (2013). High-level secretion of a recombinant protein to the culture medium with aBacillus subtilistwin-arginine translocation system inEscherichia coli. FEBS Journal [Online] 280:3810-3821. Available at: http://dx.doi.org/10.1111/febs.12376.
The twin-arginine translocation (Tat) system transports folded proteins across the plasma membrane in bacteria, and heterologous proteins can be exported by this pathway if a Tat-type signal peptide is present at the N-terminus. The system thus has potential for biopharmaceutical production in Escherichia coli, where export to the periplasm is often a favoured approach. Previous studies have shown that E. coli cells can export high levels of protein by the Tat pathway, and the protein product accummulates almost exclusively in the periplasm. In this study, we analysed E. coli cells that express the Bacillus subtilis TatAdCd system in place of the native TatABC system. We show that a heterologous model protein, comprising the TorA signal peptide linked to green fluorescent protein (TorA–GFP), is efficiently exported by the TatAdCd system. However, whereas the GFP is exported initially to the periplasm during batch fermentation, the mature protein is increasingly found in the extracellular culture medium. By the end of a 16-h fermentation, ~ 90% of exported GFP is present in the medium as active mature protein. The total protein profiles of the medium and periplasm are essentially identical, confirming that the outer membrane becomes leaky during the fermentation process. The cells are otherwise intact, and there is no large-scale release of cytoplasmic contents. Export levels are relatively high, with ~ 0.35 g GFP·L?1 culture present in the medium. This system thus offers a means of producing recombinant protein in E. coli and harvesting directly from the medium, with potential advantages in terms of ease of purification and downstream processing.
McKelvey, K., Nadappuram, B., Actis, P., Takahashi, Y., Korchev, Y., Matsue, T., Robinson, C. and Unwin, P. (2013). Fabrication, Characterization, and Functionalization of Dual Carbon Electrodes as Probes for Scanning Electrochemical Microscopy (SECM). Analytical Chemistry [Online] 85:7519-7526. Available at: http://dx.doi.org/10.1021/ac401476z.
Dual carbon electrodes (DCEs) are quickly, easily, and cheaply fabricated by depositing pyrolytic carbon into a quartz theta nanopipet. The size of DCEs can be controlled by adjusting the pulling parameters used to make the nanopipet. When operated in generation/collection (G/C) mode, the small separation between the electrodes leads to reasonable collection efficiencies of ca. 30%. A three-dimensional finite element method (FEM) simulation is developed to predict the current response of these electrodes as a means of estimating the probe geometry. Voltammetric measurements at individual electrodes combined with generation/collection measurements provide a reasonable guide to the electrode size. DCEs are employed in a scanning electrochemical microscopy (SECM) configuration, and their use for both approach curves and imaging is considered. G/C approach curve measurements are shown to be particularly sensitive to the nature of the substrate, with insulating surfaces leading to enhanced collection efficiencies, whereas conducting surfaces lead to a decrease of collection efficiency. As a proof-of-concept, DCEs are further used to locally generate an artificial electron acceptor and to follow the flux of this species and its reduced form during photosynthesis at isolated thylakoid membranes. In addition, 2-dimensional images of a single thylakoid membrane are reported and analyzed to demonstrate the high sensitivity of G/C measurements to localized surface processes. It is finally shown that individual nanometer-size electrodes can be functionalized through the selective deposition of platinum on one of the two electrodes in a DCE while leaving the other one unmodified. This provides an indication of the future versatility of this type of probe for nanoscale measurements and imaging.
Matos, C., Alanen, H., Prus, P., Uchida, Y., Freedman, R., Keshavarz-Moore, E., Robinson, C. and Ruddock, L. (2013). Efficient export of prefolded, disulfide-bonded recombinant proteins to the periplasm by the Tat pathway inEscherichia coliCyDisCo strains. Biotechnology Progress [Online] 30:281-290. Available at: http://dx.doi.org/10.1002/btpr.1858.
Numerous high-value therapeutic proteins are produced in Escherichia coli and exported to the periplasm, as this approach simplifies downstream processing and enables disulfide bond formation. Most recombinant proteins are exported by the Sec pathway, which transports substrates across the plasma membrane in an unfolded state. The Tat system also exports proteins to the periplasm, but transports them in a folded state. This system has attracted interest because of its tendency to transport correctly folded proteins, but this trait renders it unable to export proteins containing disulfide bonds since these are normally acquired only in the periplasm; reduced substrates tend to be recognized as incorrectly folded and rejected. In this study we have used a series of novel strains (termed CyDisCo) which oxidise disulfide bonds in the cytoplasm, and we show that these cells efficiently export a range of disulfide-containing proteins when a Tat signal peptide is attached. These test proteins include alkaline phosphatase (PhoA), a phytase containing four disulfide bonds (AppA), an antiinterleukin 1? scFv and human growth hormone. No export of PhoA or AppA is observed in wild-type cells lacking the CyDisCo factors. The PhoA, AppA and scFv proteins were exported in an active form by Tat in the CyDisCo strain, and mass spectrometry showed that the vast majority of the scFv protein was disulfide-bonded and correctly processed. The evidence indicates that this combination of Tat?+?CyDisCo offers a novel means of exporting active, correctly folded disulfide bonded proteins to the periplasm.
McKelvey, K., Martin, S., Robinson, C. and Unwin, P. (2013). Quantitative Local Photosynthetic Flux Measurements at Isolated Chloroplasts and Thylakoid Membranes Using Scanning Electrochemical Microscopy (SECM). Journal of Physical Chemistry B [Online] 117:7878-7888. Available at: http://dx.doi.org/10.1021/jp403048f.
Scanning electrochemical microscopy (SECM) offers a fast and quantitative method to measure local fluxes within photosynthesis. In particular, we have measured the flux of oxygen and ferrocyanide (Fe(CN)64–), from the artificial electron acceptor ferricyanide (Fe(CN)63–), using a stationary ultramicroelectrode at chloroplasts and thylakoid membranes (sourced from chloroplasts). Oxygen generation at films of chloroplasts and thylakoid membranes was detected directly during photosynthesis, but in the case of thylakoid membranes, this switched to sustained oxygen consumption at longer illumination times. An initial oxygen concentration spike was detected over both chloroplast and thylakoid membrane films, and the kinetics of the oxygen generation were extracted by fitting the experimental data to a finite element method (FEM) simulation. In contrast to previous work, the oxygen generation spike was attributed to the limited size of the plastoquinone pool, a key component in the linear electron transport pathway and a contributing factor in photoinhibition. Finally, the mobile nature of the SECM probe, and its high spatial resolution, also allowed us to detect ferrocyanide produced from a single thylakoid membrane. These results further demonstrate the power of SECM for localized flux measurements in biological processes, in this case photosynthesis, and that the high time resolution, combined with FEM simulations, allows the elucidation of quantitative kinetic information.
Ren, C., Patel, R. and Robinson, C. (2013). Exclusively membrane-inserted state of an uncleavable Tat precursor protein suggests lateral transfer into the bilayer from the translocon. FEBS Journal [Online] 280:3354-3364. Available at: http://dx.doi.org/10.1111/febs.12327.
Beck, D., Vasisht, N., Baglieri, J., Monteferrante, C., van Dijl, J., Robinson, C. and Smith, C. (2013). Ultrastructural characterisation of Bacillus subtilis TatA complexes suggests they are too small to form homooligomeric translocation pores. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research [Online] 1833:1811-1819. Available at: http://dx.doi.org/10.1016/j.bbamcr.2013.03.028.
van der Ploeg, R., Monteferrante, C., Piersma, S., Barnett, J., Kouwen, T., Robinson, C. and van Dijl, J. (2012). High-Salinity Growth Conditions Promote Tat-Independent Secretion of Tat Substrates in Bacillus subtilis. Applied and Environmental Microbiology [Online] 78:7733-7744. Available at: http://dx.doi.org/10.1128/AEM.02093-12.
The Gram-positive bacterium Bacillus subtilis contains two Tat translocases, which can facilitate transport of folded proteins across the plasma membrane. Previous research has shown that Tat-dependent protein secretion in B. subtilis is a highly selective process and that heterologous proteins, such as the green fluorescent protein (GFP), are poor Tat substrates in this organism. Nevertheless, when expressed in Escherichia coli, both B. subtilis Tat translocases facilitated exclusively Tat-dependent export of folded GFP when the twin-arginine (RR) signal peptides of the E. coli AmiA, DmsA, or MdoD proteins were attached. Therefore, the present studies were aimed at determining whether the same RR signal peptide-GFP precursors would also be exported Tat dependently in B. subtilis. In addition, we investigated the secretion of GFP fused to the full-length YwbN protein, a strict Tat substrate in B. subtilis. Several investigated GFP fusion proteins were indeed secreted in B. subtilis, but this secretion was shown to be completely Tat independent. At high-salinity growth conditions, the Tat-independent secretion of GFP as directed by the RR signal peptides from the E. coli AmiA, DmsA, or MdoD proteins was significantly enhanced, and this effect was strongest in strains lacking the TatAy-TatCy translocase. This implies that high environmental salinity has a negative influence on the avoidance of Tat-independent secretion of AmiA-GFP, DmsA-GFP, and MdoD-GFP. We conclude that as-yet-unidentified control mechanisms reject the investigated GFP fusion proteins for translocation by the B. subtilis Tat machinery and, at the same time, set limits to their Tat-independent secretion, presumably via the Sec pathway.
Monteferrante, C., Baglieri, J., Robinson, C. and van Dijl, J. (2012). TatAc, the Third TatA Subunit of Bacillus subtilis, Can Form Active Twin-Arginine Translocases with the TatCd and TatCy Subunits. Applied and Environmental Microbiology [Online] 78:4999-5001. Available at: http://dx.doi.org/10.1128/AEM.01108-12.
Two independent twin-arginine translocases (Tat) for protein secretion were previously identified in the Gram-positive bacterium Bacillus subtilis. These consist of the TatAd-TatCd and TatAy-TatCy subunits. The function of a third TatA subunit named TatAc was unknown. Here, we show that TatAc can form active protein translocases with TatCd and TatCy.
Matos, C., Branston, S., Albiniak, A., Dhanoya, A., Freedman, R., Keshavarz-Moore, E. and Robinson, C. (2012). High-yield export of a native heterologous protein to the periplasm by the tat translocation pathway in Escherichia coli. Biotechnology and Bioengineering [Online] 109:2533-2542. Available at: http://dx.doi.org/10.1002/bit.24535.
Numerous high-value recombinant proteins that are produced in bacteria are exported to the periplasm as this approach offers relatively easy downstream processing and purification. Most recombinant proteins are exported by the Sec pathway, which transports them across the plasma membrane in an unfolded state. The twin-arginine translocation (Tat) system operates in parallel with the Sec pathway but transports substrate proteins in a folded state; it therefore has potential to export proteins that are difficult to produce using the Sec pathway. In this study, we have produced a heterologous protein (green fluorescent protein; GFP) in Escherichia coli and have used batch and fed-batch fermentation systems to test the ability of the newly engineered Tat system to export this protein into the periplasm under industrial-type production conditions. GFP cannot be exported by the Sec pathway in an active form. We first tested the ability of five different Tat signal peptides to export GFP, and showed that the TorA signal peptide directed most efficient export. Under batch fermentation conditions, it was found that TorA-GFP was exported efficiently in wild type cells, but a twofold increase in periplasmic GFP was obtained when the TatABC components were co-expressed. In both cases, periplasmic GFP peaked at about the 12?h point during fermentation but decreased thereafter, suggesting that proteolysis was occurring. Typical yields were 60?mg periplasmic GFP per liter culture. The cells over-expressed the tat operon throughout the fermentation process and the Tat system was shown to be highly active over a 48?h induction period. Fed-batch fermentation generated much greater yields: using glycerol feed rates of 0.4, 0.8, and 1.2?mL?h?1, the cultures reached OD600 values of 180 and periplasmic GFP levels of 0.4, 0.85, and 1.1?g?L?1 culture, respectively. Most or all of the periplasmic GFP was shown to be active. These export values are in line with those obtained in industrial production processes using Sec-dependent export approaches. Biotechnol. Bioeng. 2012; 109: 2533–2542. © 2012 Wiley Periodicals, Inc.
Albiniak, A., Baglieri, J. and Robinson, C. (2012). Targeting of lumenal proteins across the thylakoid membrane. Journal of Experimental Botany [Online] 63:1689-1698. Available at: http://dx.doi.org/10.1093/jxb/err444.
The biogenesis of the plant thylakoid network is an enormously complex process in terms of protein targeting. The membrane system contains a large number of proteins, some of which are synthesized within the organelle, while many others are imported from the cytosol. Studies in recent years have shown that the targeting of imported proteins into and across the thylakoid membrane is particularly complex, with four different targeting pathways identified to date. Two of these are used to target membrane proteins: a signal recognition particle (SRP)-dependent pathway and a highly unusual pathway that appears to require none of the known targeting apparatus. Two further pathways are used to translocate lumenal proteins across the thylakoid membrane from the stroma and, again, the two pathways differ dramatically from each other. One is a Sec-type pathway, in which ATP hydrolysis by SecA drives the transport of the substrate protein through the membrane in an unfolded conformation. The other is the twin-arginine translocation (Tat) pathway, where substrate proteins are transported in a folded state using a unique mechanism that harnesses the proton motive force across the thylakoid membrane. This article reviews progress in studies on the targeting of lumenal proteins, with reference to the mechanisms involved, their evolution from endosymbiotic progenitors of the chloroplast, and possible elements of regulation.
Baglieri, J., Beck, D., Vasisht, N., Smith, C. and Robinson, C. (2012). Structure of TatA Paralog, TatE, Suggests a Structurally Homogeneous Form of Tat Protein Translocase That Transports Folded Proteins of Differing Diameter. Journal of Biological Chemistry [Online] 287:7335-7344. Available at: http://dx.doi.org/10.1074/jbc.M111.326355.
The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plant thylakoid membranes. Most current models for the translocation mechanism propose the coalescence of a substrate-binding TatABC complex with a separate TatA complex. In Escherichia coli, TatA complexes are widely believed to form the translocation pore, and the size variation of TatA has been linked to the transport of differently sized substrates. Here, we show that the TatA paralog TatE can substitute for TatA and support translocation of Tat substrates including AmiA, AmiC, and TorA. However, TatE is found as much smaller, discrete complexes. Gel filtration and blue native electrophoresis suggest sizes between ?50 and 110 kDa, and single-particle processing of electron micrographs gives size estimates of 70–90 kDa. Three-dimensional models of the two principal TatE complexes show estimated diameters of 6–8 nm and potential clefts or channels of up to 2.5 nm diameter. The ability of TatE to support translocation of the 90-kDa TorA protein suggests alternative translocation models in which single TatA/E complexes do not contribute the bulk of the translocation channel. The homogeneity of both the TatABC and the TatE complexes further suggests that a discrete Tat translocase can translocate a variety of substrates, presumably through the use of a flexible channel. The presence and possible significance of double- or triple-ring TatE forms is discussed.
van der Ploeg, R., Barnett, J., Vasisht, N., Goosens, V., Pother, D., Robinson, C. and van Dijl, J. (2011). Salt Sensitivity of Minimal Twin Arginine Translocases. Journal of Biological Chemistry [Online] 286:43759-43770. Available at: http://dx.doi.org/10.1074/jbc.M111.243824.
Bacterial twin arginine translocation (Tat) pathways have evolved to facilitate transport of folded proteins across membranes. Gram-negative bacteria contain a TatABC translocase composed of three subunits named TatA, TatB, and TatC. In contrast, the Tat translocases of most Gram-positive bacteria consist of only TatA and TatC subunits. In these minimal TatAC translocases, a bifunctional TatA subunit fulfils the roles of both TatA and TatB. Here we have probed the importance of conserved residues in the bifunctional TatAy subunit of Bacillus subtilis by site-specific mutagenesis. A set of engineered TatAy proteins with mutations in the cytoplasmic hinge and amphipathic helix regions were found to be inactive in protein translocation under standard growth conditions for B. subtilis or when heterologously expressed in Escherichia coli. Nevertheless, these mutated TatAy proteins did assemble into TatAy and TatAyCy complexes, and they facilitated membrane association of twin arginine precursor proteins in E. coli. Interestingly, most of the mutated TatAyCy translocases were salt-sensitive in B. subtilis. Similarly, the TatAC translocases of Bacillus cereus and Staphylococcus aureus were salt-sensitive when expressed in B. subtilis. Taken together, our present observations imply that salt-sensitive electrostatic interactions have critical roles in the preprotein translocation activity of certain TatAC type translocases from Gram-positive bacteria.
Branston, S., Matos, C., Freedman, R., Robinson, C. and Keshavarz-Moore, E. (2011). Investigation of the impact of Tat export pathway enhancement on E. coli culture, protein production and early stage recovery. Biotechnology and Bioengineering [Online] 109:983-991. Available at: http://dx.doi.org/10.1002/bit.24384.
The twin arginine translocation (Tat) pathway occurs naturally in E. coli and has the distinct ability to translocate folded proteins across the inner membrane of the cell. It has the potential to export commercially useful proteins that cannot be exported by the ubiquitous Sec pathway. To better understand the bioprocess potential of the Tat pathway, this article addresses the fermentation and downstream processing performances of E. coli strains with a wild-type Tat system exporting the over-expressed substrate protein FhuD. These were compared to strains cell-engineered to over-express the Tat pathway, since the native export capacity of the Tat pathway is low. This low capacity makes the pathway susceptible to saturation by over-expressed substrate proteins, and can result in compromised cell integrity. However, there is concern in the literature that over-expression of membrane proteins, like those of the Tat pathway, can impact negatively upon membrane integrity itself. Under controlled fermentation conditions E. coli cells with a wild-type Tat pathway showed poor protein accumulation, reaching a periplasmic maximum of only 0.5?mg?L?1 of growth medium. Cells over-expressing the Tat pathway showed a 25% improvement in growth rate, avoided pathway saturation, and showed 40-fold higher periplasmic accumulation of FhuD. Moreover, this was achieved whilst conserving the integrity of cells for downstream processing: experimentation comparing the robustness of cells to increasing levels of shear showed no detrimental effect from pathway over-expression. Further experimentation on spheroplasts generated by the lysozyme/osmotic shock method—a scaleable way to release periplasmic protein—showed similar robustness between strains. A scale-down mimic of continuous disk-stack centrifugation predicted clarifications in excess of 90% for both intact cells and spheroplasts. Cells over-expressing the Tat pathway performed comparably to cells with the wild-type system. Overall, engineering E. coli cells to over-express the Tat pathway allowed for greater periplasmic yields of FhuD at the fermentation scale without compromising downstream processing performance.
Robinson, C., Matos, C., Beck, D., Ren, C., Lawrence, J., Vasisht, N. and Mendel, S. (2011). Transport and proofreading of proteins by the twin-arginine translocation (Tat) system in bacteria. Biochimica Et Biophysica Acta-Biomembranes [Online] 1808:876-884. Available at: http://dx.doi.org/10.1016/j.bbamem.2010.11.023.
The twin-arginine translocation (Tat) system operates in plant thylakoid membranes and the plasma membranes of most free-living bacteria. In bacteria, it is responsible for the export of a number of proteins to the periplasm, outer membrane or growth medium, selecting substrates by virtue of cleavable N-terminal signal peptides that contain a key twin-arginine motif together with other determinants. Its most notable attribute is its ability to transport large folded proteins (even oligomeric proteins) across the tightly sealed plasma membrane. In Gram-negative bacteria, TatABC subunits appear to carry out all of the essential translocation functions in the form of two distinct complexes at steady state: a TatABC substrate-binding complex and separate TatA complex. Several studies favour a model in which these complexes transiently coalesce to generate the full translocase. Most Gram-positive organisms possess an even simpler “minimalist” Tat system which lacks a TatB component and contains, instead, a bifunctional TatA component. These Tat systems may involve the operation of a TatAC complex together with a separate TatA complex, although a radically different model for TatAC-type systems has also been proposed. While bacterial Tat systems appear to require the presence of only a few proteins for the actual translocation event, there is increasing evidence for the operation of ancillary components that carry out sophisticated “proofreading” activities. These activities ensure that redox proteins are only exported after full assembly of the cofactor, thereby avoiding the futile export of apo-forms. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.
Barnett, J., Robinson, C., Scanlan, D. and Blindauer, C. (2011). The Tat protein export pathway and its role in cyanobacterial metalloprotein biosynthesis. FEMS Microbiology Letters [Online] 325:1-9. Available at: http://dx.doi.org/10.1111/j.1574-6968.2011.02391.x.
Barnett, J., Lawrence, J., Mendel, S. and Robinson, C. (2011). Expression of the bifunctional Bacillus subtilis TatAd protein in Escherichia coli reveals distinct TatA/B-family and TatB-specific domains. Archives of Microbiology [Online] 193:583-594. Available at: http://dx.doi.org/10.1007/s00203-011-0699-4.
Cain, P., Holdermann, I., Sinning, I., Johnson, A. and Robinson, C. (2011). Binding of chloroplast signal recognition particle to a thylakoid membrane protein substrate in aqueous solution and delineation of the cpSRP43–substrate interaction domain. Biochemical Journal [Online] 437:149-155. Available at: http://dx.doi.org/10.1042/BJ20110270.
A cpSRP [chloroplast SRP (signal recognition particle)] comprising cpSRP54 and cpSRP43 subunits mediates the insertion of light-harvesting proteins into the thylakoid membrane. We dissected its interaction with a full-length membrane protein substrate in aqueous solution by insertion of site-specific photo-activatable cross-linkers into in vitro-synthesized Lhcb1 (major light-harvesting chlorophyll-binding protein of photosystem II). We show that Lhcb1 residues 166–176 cross-link specifically to the cpSRP43 subunit. Some cross-link positions within Lhcb1 are in the ‘L18’ peptide required for targeting of cpSRP substrates, whereas other cross-linking positions define a new targeting signal in the third transmembrane span. Lhcb1 was not found to cross-link to cpSRP54 at any position, and cross-linking to cpSRP43 is unaffected by the absence of cpSRP54. cpSRP43 thus effectively binds substrates autonomously, and its ability to independently bind an extended 20+-residue substrate region highlights a major difference with other SRP types where the SRP54 subunit binds to hydrophobic target sequences. The results also show that cpSRP43 can bind to a hydrophobic, three-membrane span, substrate in aqueous solution, presumably reflecting a role for cpSRP in the chloroplast stroma. This mode of action, and the specificity of the cpSRP43–substrate interaction, may be associated with cpSRP's unique post-translational mode of action.
Vladimirou, E., Li, M., Aldridge, C., Frigerio, L., Kirkilionis, M. and Robinson, C. (2009). Diffusion of a membrane protein, Tat subunit Hcf106, is highly restricted within the chloroplast thylakoid network. FEBS Letters [Online] 583:3690-3696. Available at: http://dx.doi.org/10.1016/j.febslet.2009.10.057.
The thylakoid membrane forms stacked thylakoids interconnected by ‘stromal’ lamellae. Little is known about the mobility of proteins within this system. We studied a stromal lamellae protein, Hcf106, by targeting an Hcf106-GFP fusion protein to the thylakoids and photobleaching. We find that even small regions fail to recover Hcf106-GFP fluorescence over periods of up to 3 min after photobleaching. The protein is thus either immobile within the thylakoid membrane, or its diffusion is tightly restricted within distinct regions. Autofluorescence from the photosystem II light-harvesting complex in the granal stacks likewise fails to recover. Integral membrane proteins within both the stromal and granal membranes are therefore highly constrained, possibly forming ‘microdomains’ that are sharply separated.
Cain, P., Hall, M., Schröder, W., Kieselbach, T. and Robinson, C. (2009). A novel extended family of stromal thioredoxins. Plant Molecular Biology [Online] 70:273-281. Available at: http://dx.doi.org/10.1007/s11103-009-9471-4.
Thioredoxins play key regulatory roles in chloroplasts by linking photosynthetic light reactions to a series of plastid functions. In addition to the established groups of thioredoxins, f, m, x, and y, novel plant thioredoxins were also considered to include WCRKC motif proteins, CDSP32, the APR proteins, the lilium proteins and HCF164. Despite their important roles, the subcellular locations of many novel thioredoxins has remained unknown. Here, we report a study of their subcellular location using the cDNA clone resources of TAIR. In addition to filling all gaps in the subcellular map of the established chloroplast thioredoxins f, m, x and y, we show that the members of the WCRKC family are targeted to the stroma and provide evidence for a stromal location of the lilium proteins. The combined data from this and related studies indicate a consistent stromal location of the known Arabidopsis chloroplast thioredoxins except for thylakoid-bound HCF164.
Warren, G., Oates, J., Robinson, C. and Dixon, A. (2009). Contributions of the Transmembrane Domain and a Key Acidic Motif to Assembly and Function of the TatA Complex. Journal of Molecular Biology [Online] 388:122-132. Available at: http://dx.doi.org/10.1016/j.jmb.2009.02.060.
The twin-arginine translocase (Tat) pathway transports folded proteins across bacterial and thylakoid membranes. In Escherichia coli, a membrane-bound TatA complex, which oligomerizes to form complexes of less than 100 to more than 500 kDa, is considered essential for translocation. We have studied the contributions of various TatA domains to the assembly and function of this heterogeneous TatA complex. The TOXCAT assay was used to analyze the potential contribution of the TatA transmembrane (TM) domain. We observed relatively weak interactions between TatA TM domains, suggesting that the TM domain is not the sole driving force behind oligomerization. A potential hydrogen-bonding role for a TM domain glutamine was also investigated, and it was found that mutation blocks transport at low expression levels, while assembly is unaffected at higher expression levels. Analysis of truncated TatA proteins instead highlighted an acidic motif directly following the TatA amphipathic helix. Mutating these negatively charged residues to apolar uncharged residues completely blocks activity, even at high levels of TatA, and appears to disrupt ordered complex formation.
Barnett, J., van der Ploeg, R., Eijlander, R., Nenninger, A., Mendel, S., Rozeboom, R., Kuipers, O., van Dijl, J. and Robinson, C. (2009). The twin-arginine translocation (Tat) systems from Bacillus subtilis display a conserved mode of complex organization and similar substrate recognition requirements. FEBS Journal [Online] 276:232-243. Available at: http://dx.doi.org/10.1111/j.1742-4658.2008.06776.x.
The twin arginine translocation (Tat) system transports folded proteins across the bacterial plasma membrane. In Gram-negative bacteria, membrane-bound TatABC subunits are all essential for activity, whereas Gram-positive bacteria usually contain only TatAC subunits. In Bacillus subtilis, two TatAC-type systems, TatAdCd and TatAyCy, operate in parallel with different substrate specificities. Here, we show that they recognize similar signal peptide determinants. Both systems translocate green fluorescent protein fused to three distinct Escherichia coli Tat signal peptides, namely DmsA, AmiA and MdoD, and mutagenesis of the DmsA signal peptide confirmed that both Tat pathways recognize similar targeting determinants within Tat signals. Although another E. coli Tat substrate, trimethylamine N-oxide reductase, was translocated by TatAdCd but not by TatAyCy, we conclude that these systems are not predisposed to recognize only specific Tat signal peptides, as suggested by their narrow substrate specificities in B. subtilis. We also analysed complexes involved in the second Tat pathway in B. subtilis, TatAyCy. This revealed a discrete TatAyCy complex together with a separate, homogeneous, ? 200 kDa TatAy complex. The latter complex differs significantly from the corresponding E. coli TatA complexes, pointing to major structural differences between Tat complexes from Gram-negative and Gram-positive organisms. Like TatAd, TatAy is also detectable in the form of massive cytosolic complexes.
Matos, C., Di Cola, A. and Robinson, C. (2009). TatD is a central component of a Tat translocon-initiated quality control system for exported FeS proteins in Escherichia coli. EMBO Reports [Online] 10:474-479. Available at: http://dx.doi.org/10.1038/embor.2009.34.
Bacterial Tat systems export folded proteins, including FeS proteins such as NrfC and NapG, which acquire their cofactors before translocation. NrfC and NapG are proofread by the Tat pathway, and misfolded examples are degraded after interaction with the translocon. Here, we identify TatD as a crucial component of this quality control system in Escherichia coli. NrfC/NapG variants lacking FeS centres are rapidly degraded in wild-type cells but stable in a DeltatatD strain. The precursor of another substrate, FhuD, is also transiently detected in wild-type cells but stable in the DeltatatD strain. Surprisingly, these substrates are stable in DeltatatD cells that overexpress TatD, and export of the non-mutated precursors is inhibited. We propose that TatD is part of a quality control system that is intimately linked to the Tat export pathway, and that the overexpression of TatD leads to an imbalance between the two systems such that both Tat-initiated turnover and export are prevented.
Aldridge, C., Cain, P. and Robinson, C. (2009). Protein transport in organelles: Protein transport into and across the thylakoid membrane. FEBS Journal [Online] 276:1177-1186. Available at: http://dx.doi.org/10.1111/j.1742-4658.2009.06875.x.
The chloroplast thylakoid is the most abundant membrane system in nature, and is responsible for the critical processes of light capture, electron transport and photophosphorylation. Most of the resident proteins are imported from the cytosol and then transported into or across the thylakoid membrane. This minireview describes the multitude of pathways used for these proteins. We discuss the huge differences in the mechanisms involved in the secretory and twin-arginine translocase pathways used for the transport of proteins into the lumen, with an emphasis on the differing substrate conformations and energy requirements. We also discuss the rationale for the use of two different systems for membrane protein insertion: the signal recognition particle pathway and the so-called spontaneous pathway. The recent crystallization of a key chloroplast signal recognition particle component provides new insights into this rather unique form of signal recognition particle.