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Biochimica et Biophysica Acta 1459 (2000) 325^330 www.elsevier.com/locate/bba A novel protein transport system involved in the biogenesis of bacterial electron transfer chains Ben C. Berks a; *, Frank Sargent a , Erik De Leeuw a , Andrew P. Hinsley a , Nicola R. Stanley a;b , Rachael L. Jack a;b , Grant Buchanan b , Tracy Palmer a;b a Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK b Department of Molecular Microbiology, John Innes Centre, Colney, Norwich, NR4 7UH, UK Received 15 May 2000; accepted 12 June 2000 Abstract The Tat system is a recently discovered bacterial protein transport pathway that functions primarily in the biosynthesis of proteins containing redox active cofactors. Analogous transport systems are found in plant organelles. Remarkably and uniquely the Tat system functions to transported a diverse range of folded proteins across a biological membrane, a feat that must be achieved without rendering the membrane freely permeable to protons and other ions. Here we review the operation of the bacterial Tat system and propose a model for the structural organisation of the Tat preprotein translocase. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Protein transport; Redox protein; Metalloprotein biosynthesis; Tat pathway; Escherichia coli 1. Introduction In bacteria the generation of energy by respiratory or photosynthetic electron transfer chains involves the cytoplasmic membrane. In order to allow protonmotive force generation by the redox loop mechanism, and to avoid the energetic costs of transporting substrate molecules (electron donors or terminal electron acceptors) obtained from the extracellular environment into the cytoplasm, a proportion of the redox active components of the electron transfer chains are always located at the extracellular side of this membrane. Thus bacterial growth in most environments depends upon the bacterium being able to * Corresponding author. Fax: +44 (1603) 592250; E-mail: [email protected] produce cofactor-containing proteins in the extracellular compartment. Biosynthesis of such extracytoplasmic redox proteins must involve both protein transport across the cytoplasmic membrane and cofactor insertion into the protein. However, combining these two processes presents a severe biosynthetic problem to the cell. To understand why this is we need to consider how a protein without a cofactor is normally exported from the bacterial cytoplasm. In Gram-negative bacteria, such as Escherichia coli, the extracellular compartment is the periplasm. Proteins are normally exported to the periplasm, or to the periplasmic side of the cytoplasmic membrane, by the Sec apparatus [1]. Proteins destined for the Sec pathway are usually synthesised as precursor proteins with N-terminal signal peptides that direct the protein to the Sec system, and that are proteolytically removed from the protein following the trans- 0005-2728 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 5 - 2 7 2 8 ( 0 0 ) 0 0 1 6 8 - 7 BBABIO 44923 7-8-00 326 B.C. Berks et al. / Biochimica et Biophysica Acta 1459 (2000) 325^330 port event. Transport by the Sec system is energised by a combination of ATP hydrolysis and the transmembrane proton electrochemical gradient. Prior to transport the substrate protein is kept in an extended conformation. The protein is then threaded through the Sec preprotein translocase and only when it reaches the periplasm does the protein fold. The problem that biosynthesis of cofactor-containing periplasmic proteins encounters is the requirement of the Sec apparatus for an essentially unstructured precursor protein. In order to stably bind cofactor the protein must fold, yet if the protein is folded it is no longer an acceptable substrate for the Sec pathway. One possible way to circumvent this problem is to transport protein and cofactor across the cytoplasmic membrane separately, and then carry out cofactor insertion in the periplasm. This is the solution adopted for proteins with some types of cofactor, primarily those of c-type cytochromes (see article by Tho«ny-Meyer in this issue [2]). However, for periplasmic proteins containing most other types of cofactor, including iron-sulfur clusters, molybdopterin cofactors and nucleotide cofactors, the bacterium allows cofactor insertion in the cytoplasm. The cofactor-containing precursor is then targeted to an export apparatus that is completely distinct from the Sec system [3] and that has the remarkably property of being able to transport a folded proteins across a biological membrane without rendering the membrane freely permeable to protons and other ions. This Sec-independent protein transport pathway is usually called the Tat (twin arginine translocation) system [4] because targeting to the pathway involves signal peptides containing two consecutive and invariant arginine residues. An alternative Mtt designation has also been employed [5]. Here we brie£y review the major features of the bacterial Tat transport pathway as they are currently understood and propose a model for the structural organisation of the components of the Tat preprotein translocase. More detailed reviews of the Tat pathway can be found in [6^8]. It has recently become apparent that the bacterial Tat system is structurally and mechanistically analogous to the vpH-dependent thylakoid import pathway of plant chloroplasts [9^13]. In addition, the mitochondrial genomes of all higher plants encode a protein homologous to one component of the bac- terial Tat pathway suggesting that the mitochondria of plants also possess a Tat-like protein transport system [5,14]. 2. Targeting to the Tat pathway The signal peptides directing proteins to the Tat export pathway have a tripartite structure resembling that of Sec signal peptides. A basic (n-) region is followed by a hydrophobic (h-) region and then a c-region containing the recognition site for the protease that will remove the signal peptide following transport. However, several features distinguish Tat and Sec signal peptides. While Sec signal peptides show no amino acid sequence conservation, Tat signal peptides have a consensus Ser-Arg-Arg-Xaa-PheLeu-Lys amino acid sequence motif (with Xaa representing a polar amino acid) at the n-region/h-region boundary [8,15]. Within the Tat consensus motif the arginine residues are invariant and are normally essential for transport by the Tat pathway [15^18]. Recent data, however, indicate that in certain signal peptide contexts conservative substitution of a lysine for one of the arginine residues still allows weak Tat targeting activity [15]. The non-arginine residues of the consensus motif are each present in at least 50% of Tat signal peptides. Mutating these non-arginine consensus residues, in particular substitutions at the consensus phenylalanine position, can lead to severe defects in Tat transport suggesting that the entire consensus motif is important for Tat targeting [15]. In addition to the presence of a consensus motif, Tat signal peptides di¡er from Sec signal peptides in possessing a substantially less hydrophobic h-region [17]. The reduced hydrophobicity of Tat signal peptide hregions has been demonstrated to be critical both for targeting to the Tat pathway and for avoiding e¤cient interaction with the Sec machinery [17]. Tat signal peptides often contain basic residues in the c-region [11]. Recent mutagenesis data suggest that the presence of, or sign of, the charge in the c-region of Tat signal peptides is not a major determinant in targeting to the Tat pathway [15]. However, positively charged residues are not normally found in the vicinity of the signal peptide processing site of Sec signal peptides, and when such residues are introduced experimentally transport e¤ciency is re- BBABIO 44923 7-8-00 B.C. Berks et al. / Biochimica et Biophysica Acta 1459 (2000) 325^330 duced (e.g. [19]). It is thus possible that the positively charged c-regions found in many Tat signal peptides are an additional determinant that prevents the signal peptide functionally interacting with the Sec pathway [17]. A ¢nal di¡erence between Tat and Sec signal peptide is their length. Sec signal peptides generally comprise no more that 26 amino acids while Tat signal peptides are normally 26^60 amino acids long [8]. The additional length of Tat signal peptides is mainly due to their larger n-regions [17]. The functional signi¢cance of the greater length of Tat signal peptides is currently unclear. Tat signal peptides undoubtedly act as Tat-speci¢c targeting determinants but could they have additional functions? It is likely that the cell has a mechanism to prevent substrate proteins accessing the Tat pathway until cofactor insertion has been completed or, in the case of hetero-oligomeric proteins exported using a single signal peptide, a multisubunit complex has been formed [3,6]. The most obvious way to achieve such control of transport is for the signal peptide to be sequestered by speci¢c protein-protein interactions, either with conformations of the substrate protein that are no longer available in the fully folded mature protein, or by protein-speci¢c chaperones that can sense the folding state of the substrate protein. A particularly attractive idea is that the putative signal peptide-binding chaperones could be the same proteins that are involved in, and are therefore released following, cofactor insertion. 3. Tat pathway components Studies in E. coli have identi¢ed two operons, tatABCD and tatE, coding for components of the Tat pathway (Fig. 1). Strains with in-frame deletions in tatB, in tatC, or in a combination of both tatA and tatE, are completely defective in the translocation of precursor proteins with twin-arginine signal peptides [4,14,20]. Consistent with a role in transmembrane protein translocation these four genes encode integral membrane proteins [21]. TatA and TatE are more than 50% identical in sequence and both in turn exhibit weak sequence similarity to TatB. The TatA/B/ E proteins have a predicted structure in which an Nterminal transmembrane helix is followed at the cytoplasmic side of the plasma membrane by an am- 327 Fig. 1. Organisation of the E. coli tat operons. A putative stem-loop structure lies between the tatC and tatD genes. Arrows indicate transcripts associated with the tatA locus [21]. phipathic helix and then a sequence-variable region. TatC is predicted to contain six transmembrane helices with no extensive extramembranous domains. It has recently been demonstrated that neither the water-soluble tatD gene product, nor two other E. coli genes encoding TatD homologues are required for protein export [21]. Genomic analysis of a wide range of bacterial species suggests that a minimum of two copies of TatA/ B/E-like proteins and one copy of a TatC-like protein is required to form a functional Tat system [6,7]. Recent data suggest that the plant thylakoid vpHdependent transport system also utilises at least two TatA/B/E-type proteins [22]. 4. A model for the structural organisation of the Tat preprotein translocase Since the essential Tat components are all integral membrane proteins we speculate that these proteins come together to form the Tat preprotein translocase. Fig. 2 depicts a model for the organisation of the Tat proteins within the translocase complex. The model was formulated on the basis of the arguments outlined in this section. We reasoned that the translocase must, at some stage within the transport cycle, provide a water¢lled channel across the membrane through which the folded substrate proteins are transported. Since the structures of many Tat substrates are known, we can deduce that this channel must attain a diameter BBABIO 44923 7-8-00 328 B.C. Berks et al. / Biochimica et Biophysica Acta 1459 (2000) 325^330 î when fully open [6]. The channel of at least 60 A should be surrounded by proteinaceous elements, presumably membrane-spanning K-helices. The number of transmembrane helices required to form a î is considerpore with an internal diameter of 60 A ably greater than the number of predicted transmembrane helices in the known Tat components [6]. This suggests that the channel must be formed from multiple copies of at least one Tat subunit. We have recently co-puri¢ed E. coli TatA and TatB in detergent solution as a complex with a molecular mass of around 600 kDa (F. Sargent, T. Palmer and B.C. Berks, unpublished observations). The mass of this complex is similar to that inferred for the plant vpHdependent pathway translocase [23]. TatA is present in a large molar excess over TatB in the puri¢ed E. coli TatAB complex. We therefore suggest that TatA is the major channel-forming subunit of the Tat translocase. TatE is functionally interchangeable with TatA [4,20] and so would presumably also have a channel forming function. However, our puri¢cation of an exclusively TatAB-containing complex suggests that there are probably separate TatA and TatE based translocases. In addition to providing a transport channel for the substrate, the translocase must have a mechanism for identifying that substrate. Since targeting to the Tat pathway involves the recognition of a highly conserved amino acid sequence motif within precursor signal peptides it is likely that the translocase makes very speci¢c protein-protein interactions with the Tat signal peptide. We guess that the amino acids in the translocase that form the binding site for the twin-arginine consensus motif should be strongly conserved during evolution. TatA/B/E family proteins show only limited sequence conservation, with a glycine residue at the junction between the predicted transmembrane and amphipathic helices the only invariant residue. We therefore think it unlikely that these proteins provide the signal peptide binding site. In contrast, TatC proteins show substantial sequence conservation, and so we have proposed that TatC proteins contain the site of signal peptide recognition [6]. Note that a single copy of the TatC subunit per translocase would be su¤cient to accomplish the proposed signal peptide binding function. In the model shown in Fig. 2 TatB is assigned the role of physically connecting, and thus mediating communication between, the TatA and TatC subunits. We know from our puri¢cation of a TatAB complex that the TatA and TatB subunits must physically interact. Since TatB has overall structural Fig. 2. A model for the structural organisation of the Tat preprotein translocase of E. coli. A cross-section through the translocase is depicted. A substrate protein is shown bound to TatC by its signal sequence. BBABIO 44923 7-8-00 B.C. Berks et al. / Biochimica et Biophysica Acta 1459 (2000) 325^330 similarity to TatA we reason that TatB could act as a pseudo-TatA subunit and integrate itself into the TatA channel structure. We also know that the TatC protein is completely undetectable in a strain unable to produce TatB suggesting that TatB stabilises, and thus makes protein-protein interactions with, TatC [20]. Further, we know that increasing the ratio of TatB molecules to other Tat components prevents the Tat system from functioning [20]. A possible interpretation of this observation that is consistent with the TatB function proposed here is that, as the concentration of TatB in the cell increases, the TatB molecules bound to the TatC subunits are no longer also bound to TatA subunits. Thus the physical connection between precursor binding site and channel is broken. 5. Bioenergetics The mechanism of the Tat translocase is constrained in two ways by the proton electrochemical gradient (vp) across the cytoplasmic membrane. First, by analogy to the vpH-dependent thylakoid transporter, protein translocation by the bacterial Tat translocase is likely to be energised exclusively by vp, and the e¡ects of uncouplers on Tat transport in whole bacterial cells are consistent with this idea [3,17,18]. However, nothing is currently know about the molecular basis of the coupling of protein transport in the Tat system to vp. Second, the mechanism of protein translocation by the Tat system must prevent substantive ion leak across the cytoplasmic membrane during the transport process to avoid uncoupling the cell. This constraint may prevent mechanisms which envisage a dynamic pore that packs around the substrate protein since it is hard to see how such packing could be su¤ciently tight to prevent the movement of protons. Instead, it is more likely that the translocase has two protonimpermeable gates that are never open simultaneously. Clearly our studies of this remarkable and mechanistically unique protein transport pathway are at an early stage. A full understanding of the Tat system will not only be of high intellectual interest but will be fundamental to our understanding of the biogenesis of biological electron transport chains. 329 Acknowledgements We wish to thank the many colleagues with whom we have discussed aspects of the Tat system. Work in the authors' laboratories is supported by the Biotechnology and Biological Sciences Research Council, the Royal Society, the University of East Anglia and the Commission of the European Community. References [1] A.P. Pugsley, The complete general-secretory pathway in Gram-negative bacteria, Microbiol. Rev. 57 (1993) 50^108. [2] Tho«ny-Meyer, Haem-polypeptide interactions during cytochrome c maturation, Biochem. Biophys. Acta 1459 (2000) 315^323 (this issue). [3] C.-L. Santini, B. Ize, A. Chanal, M. Mu«ller, G. Giordano, L.-F. Wu, A novel Sec-independent periplasmic protein translocation pathway in Escherichia coli, EMBO J. 17 (1998) 101^112. [4] F. Sargent, E. Bogsch, N.R. Stanley, M. Wexler, C. Robinson, B.C. Berks, T. Palmer, Overlapping functions of components of a bacterial Sec-independent protein export pathway, EMBO J. 17 (1998) 3640^3650. [5] J.H. Weiner, P.T. Bilous, G.M. Shaw, S.P. Lubitz, L. Frost, G.H. Thomas, J.A. Cole, R.J. Turner, A novel and ubiquitous system for membrane targeting and secretion of cofactor-containing proteins, Cell 93 (1998) 93^101. [6] B.C. Berks, F. Sargent, T. Palmer, The Tat protein export pathway, Mol. Microbiol. 35 (2000) 260^274. [7] L.-F. Wu, B. Ize, A. Chanal, Y. Quentin, G. Fichant, Bacterial twin-arginine signal peptide-dependent protein translocation pathway: evolution and mechanism, J. Mol. Microbiol. Biotechnol. 2 (2000) 179^189. [8] B.C. Berks, A common export pathway for proteins binding complex redox cofactors?, Mol. Microbiol. 22 (1996) 393^ 404. [9] A.M. Settles, A. Yonetani, A. Baron, D.R. Bush, K. Cline, R. Martienssen, Sec-independent protein translocation by the maize Hcf106 protein, Science 278 (1997) 1467^1470. [10] H. Mori, K. Cline, A signal peptide that directs non-Sec transport in bacteria also directs e¤cient and exclusive transport on the thylakoid delta pH pathway, J. Biol. Chem. 273 (1998) 11405^11408. [11] M. Wexler, E. Bogsch, R.B. Klo«sgen, T. Palmer, C. Robinson, B.C. Berks, Targeting signals for a bacterial Sec-independent export system direct plant thylakoid import by the vpH pathway, FEBS Lett. 431 (1998) 339^342. [12] K. Keegstra, K. Cline, Protein import and routing systems of chloroplasts, Plant Cell 11 (1999) 557^570. [13] C. Robinson, this issue. [14] E. Bogsch, F. Sargent, N.R. Stanley, B.C. Berks, C. Robinson, T. Palmer, An essential component of a novel bacterial BBABIO 44923 7-8-00 330 [15] [16] [17] [18] B.C. Berks et al. / Biochimica et Biophysica Acta 1459 (2000) 325^330 protein export system with homologues in plastids and mitochondria, J. Biol. 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Biochem. 263 (1999) 543^551. [19] P. Li, J. Beckwith, H. Inouye, Alteration of the amino terminus of the mature sequence of a periplasmic protein can severely a¡ect protein export in Escherichia coli, Proc. Natl. Acad. Sci. USA 85 (1988) 7685^7689. [20] F. Sargent, N.R. Stanley, B.C. Berks, T. Palmer, Sec-independent protein translocation in Escherichia coli: a distinct and pivotal role for the TatB protein, J. Biol. Chem. 274 (1999) 36073^36082. [21] M. Wexler, F. Sargent, R.L. Jack, N.R. Stanley, E. Bogsch, C. Robinson, B.C. Berks, B.C., T. Palmer, TatD is a cytoplasmic protein with DNase activity. No requirement for TatD family proteins in Sec-independent protein export, J. Biol. Chem. 275 (2000) 16717^16722. [22] H. Mori, E.J. Summer, X. Ma, K. Cline, Component speci¢city for the thylakoid Sec and delta pH-dependent protein transport pathways, J. Cell Biol. 146 (1999) 45^55. [23] J. Bergho«fer, R.B. Klo«sgen, Two distinct translocation intermediates can be distinguished during protein transport by the TAT (vpH) pathway across the thylakoid membrane, FEBS Lett. 460 (1999) 328^332. BBABIO 44923 7-8-00