2.A.64 The Twin Arginine Targeting (Tat) Family
The TatABCE system of E. coli has been both sequenced and functionally characterized (Berks, 1996; Berks et al., 2000a,b). This system forms a large (~600 kDa) complex which interacts with fully folded redox proteins that have an N-terminal S/TRRXFLK 'twin arginine' leader motif (Sargent et al., 2001). It translocates several redox enzymes to the E. coli periplasm including nitrate reductase (NapA) and trimethylamine N-oxide reductase (TorA) which have this leader motif (Bogsch et al., 1998; Santini et al., 1998; Sargent et al., 1998). Hydrogenases, formate dehydrogenases and several non-redox proteins (about two dozen in E. coli), including virulence factors, periplasmic binding proteins, and enzymes involved in envelope biogenesis, have the 'twin R' motif and probably use this pathway. Among these substrate enzyme complexes are several that contain integral membrane proteins (Sargent et al., 2002). These proteins associate with their cofactors in the cell cytoplasm before translocation. TatABCE is also required for the assembly of the E. coli dimethyl sulfoxide reductase (DmsABC) (Weiner et al., 1998; Sambasivarao et al., 2001). The Tat system functions independently of other types of protein secretory systems present in E. coli. However, Tat signal sequences exhibit overlap with Sec signal sequences and can direct proteins to both secretory systems (Tullman-Ercek et al., 2007; Kreutzenbeck et al., 2007). One report suggests that Tat systems can transport small unstructured hydrophilic proteins although hydrophobic patches can abort transport at a late stage (Richter et al., 2007). Fan et al. (2010) have reputed that one signal is sufficient for the stepwise transport of two distinct passenger proteins across the thylakoid membrane. The 2012 view of how the Tat system works is provided in (Palmer and Berks 2012). The pore-forming part of TatA has been resolved by NMR(Walther et al. 2010). TatC helix 5 and the TatB transmembrane helix interact (Kneuper et al. 2012) and cooperate to produce recognition of Tat signal peptides in E. coli (Lausberg et al. 2012). Transmembrane insertion of Tat peptides is driven by TatC and regulated by TatB (Fröbel et al. 2012). In the TatBC receptor complex, the transmembrane helix of each TatB is sandwiched between two TatCs, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. TatA also associates with TatC at the polar cluster site (Alcock et al. 2016).
The TatA, TatB, TatC and TatE proteins have 1, 1, 6 and 1 putative transmembrane α-helical spanners (TMSs), respectively (sizes of 98, 171, 258 and 67 amino acyl residues, respectively) (Punginelli et al., 2007). TatA, TatB and TatE are homologous, and TatA and TatE, which are more similar to each other than they are to TatB, can partially substitute for each other and form heterooligomers (Eimer et al. 2015). Both TatA and TatB have the N- out and C- in orientation (Koch et al. 2012). They can be mutationally modified so only one of these proteins is required (Barrett et al., 2007). The transmembrane and amphipathic helical regions of TatA, B and E are critical for function, but their C-terminal domains are not (Lee et al., 2002). Chan et al. (2007) have reported that the N-terminus of TatA is located in the cytoplasm rather than the periplasm. The C-terminus might have a dual topology; its orientation in the membrane could be dependent on the membrane potential. Thus, two architectures of TatA may exist in the membrane: one with a single transmembrane helix and the other with two transmembrane helices. The double transmembrane helix topology might be the building block for the translocation channel. However this suggestion was not supported by more recent work (Koch et al. 2012). Multimeric TatA may form an expandable protein-conducting channel (Lange et al., 2007 ). The NMR solution structure of TatA has been published (Hu et al., 2010). Structural and biophysical studies of the amphipathic α-helical region of TatA from E. coli have been conducted (Chan et al., 2011). TatB functions as an oligomeric binding site for folded Tat precursor proteins (Maurer et al. 2010).
TatC is required for interaction of TatA with TatB (Bolhuis et al., 2001) and has 6 established TMSs with both the N- and C-termini in the cytoplasm (Drew et al., 2002). Interaction and assembly of the substrate protein with the Tat complex appears to occur in several steps (Alami et al., 2003). First, the twin arginine precursor associates with TatC. Second, TatB associates with TatC. Third, TatA association occurs only in the presence of a transmembrane pH gradient. The TatA/B protein-translocating complex channel, of variable size, accommodates and transports the substrate protein complex (Dabney-Smith, 2006; Gerard and Cline, 2006; Gohlke et al., 2005; Hicks et al., 2005). TatB may mediate transfer of the folded substrate from TatC to the Tat pore (Alami et al., 2003). TatC may be peripheral to the TatA/B/C channel which accomodates proteins via the hydrophilic lining of amphipathic α-helices (Greene et al., 2007). TatB and TatC both recognize the twin arginine signal sequence (Strauch and Georgiou, 2007), and TatB thus forms an oligomeric binding
site that transiently accomodates folded Tat precursors (Maurer et al., 2010). A TatC dimer is probably at the core of the Tat complex (Maldonado et al., 2011).
Gram-negative bacteria with fully sequenced genomes exhibit only a single TatC homologue, but many Gram-positive bacteria and archaea encode two (Yen et al., 2002). Gram-positive bacteria such as Bacillus subtilis have two independently functioning systems (TatAyCy and TatAdCd in B. subtilis). Expression of the bifunctional B. subtilis TatAd protein in E. coli revealed distinct TatA/B-family and TatB-specific domains (Barnett et al., 2011). Plants have both mitochondrial and chloroplast homologues; for example, Arabidopsis thaliana has one chloroplast TatC homologue and two TatA homologues as well as two putative mitochondrial TatC homologues. These proteins are not found in yeast and animals. A few bacteria (i.e., Rickettsia prowazekii) have only one TatA homologue, but most have two, and several have three (α-proteobacteria and Bacillus subtilis). There are usually two sequence dissimilar paralogues (e.g., TatA and TatB in E. coli) and sometimes one sequence similar paralogue (e.g., TatE) (Yen et al., 2002).
The energetics of the chloroplast Tat system (Alder and Theg, 2003; Berks et al., 2005; Theg et al., 2005) suggest a protein:H+ antiport mechanism with about 100,000 H+ released per transported protein (equivalent to about 104 ATP). The Tat pathway may use about 3% of the total chloroplast energy yield. In chloroplasts, cptatC and Hcf106 form a signal peptide precursor-bound receptor complex which assembles the oligomeric Tha4 translocation pore (Dabney-Smith et al., 2006; Gérard and Cline, 2006). A 'trap door' mechanism was proposed in which oligomers of Tha4 amphipathic helices fold into the membrane to allow form-fitting passage of the substrate precursor protein (Dabney-Smith et al., 2006). After translocation, the complex dissociates (Gérard and Cline, 2006). Tha4 oligomers may dock with a precursor-receptor complex and undergo a conformational switch that results in activation for protein transport. This possibly involves accretion of additional Tha4 subunits into a larger transport-active homo-oligomer (Dabney-Smith and Cline, 2009). Multiple precursor proteins bound to a single receptor complex can be transported together (Ma and Cline, 2010).
Three components are required for Tat transport, cpTatC, Hcf106, and Tha4, in thylakoids (the orthologous TatC, TatB, and TatA, respectively, in bacteria). The thylakoid Tat system has been experimentally staged into several steps. (1) The precursor protein binds to a cpTatC-Hcf106 receptor complex, (2) a Tha4 oligomer assembles with the precursor-receptor complex to form the putative translocase, (3) and the precursor is transported into the lumen. After transport, Tha4 dissociates from the receptor complex, resetting the system for another round of transport (Gérard and Cline, 2007). Tha4 has been shown to undergo conformational changes that accompany protein transport (Aldridge et al. 2012).
A genomic survey indicates that the TAT pathway is utilized to varying extents depending on the bacterium, from 0 to 20% of the total secreted proteins (Dilks et al., 2003). While many prokaryotes use it primarily for the secretion of redox protein complexes, some Gram-positive and Gram-negative bacteria as well as archaea, use it to export non-redox proteins. The composition of the TAT protein complex does not correlate with numbers of substrates but does with organismal phylogeny (Dilks et al., 2003). One report suggests that the TAT pathway can export outer membrane proteins without a cleavable signal sequence (Ferrandez and Condemine, 2008). Streptomyces species have TatA, B and C (Yen et al., 2002) and translocate large numbers of lipoproteins out via the Tat pathway. Lipoprotein biogenesis is essential in S. coelicolor (Thompson et al., 2010).
TatC has been shown to serve as a specificity determinant for protein secretion via the Tat system (Jongbloed et al., 2000). However, other proteins may play a role in recognition of the 'twin arginine' motif (Oresnik et al., 2001). For example, DmsD is a Tat leader binding protein that interacts with TatB and TatC (Papish et al., 2003). The energy-coupling mechanism for transport involves the pmf in both chloroplasts and E. coli (Dalbey and Robinson, 1999; Settles et al., 1997; 1998). The E. coli TatA may be capable of flipping orientation in the membrane so that its C-terminus is either in the cytoplasm or in the periplasm (Gouffi et al., 2004), but the physiological significance of this observation is not known.
The heteromultimeric TatAd/TatCd of B. subtilis has been studied with respect to the functions of its two subunits (Schreiber et al., 2006). TatAd localizes to the cytosol or membrane. Soluble TatA can bind to the twin arg signal peptide of pre-PhoD prior to membrane integration. It recruits the substrate protein to the membrane by interaction with TatC. TatC (1) facilitates the membrane association of TatA and (2) stabilizes it (Schreiber et al., 2006). B. subtilis has two Tat complexes, each with two Tat subunits, TatA and TatC. TatAdCd and TatAyCy transport different substrate proteins (Barnett et al., 2009). PhoD is secreted by the TatAdCd complex whereas YwbN is secreted by the TatAyCy complex (Eijlander et al., 2009).
The Tat systems in most Gram-positive bacteria consists of TatA and TatC. TatA is a bifunctional subunit, which can form a protein-conducting channel by self-oligomerization and can also participate in substrate recognition. Hu et al. (2010) reported the solution structure of the TatA(d) protein from Bacillus subtilis by NMR spectroscopy, the first structure of the Tat system at atomic resolution. TatA(d) shows an L-shaped structure formed by a transmembrane helix and an amphipathic helix, while the C-terminal tail is largely unstructured. These results support the postulated topology of TatA(d) in which the transmembrane helix is inserted into the lipid bilayer while the amphipathic helix lies at the membrane-water interface. Moreover, the structure of TatA(d) revealed the structural importance of several conserved residues at the hinge region, thus shedding new light on further elucidation of the protein transport mechanism. The Tat system in Streptomyces species functions in the assembly of the cytochrome bc1 complex (Hopkins et al. 2013).
In E. coli, substrate proteins initially bind to the integral membrane TatBC complex which then recruits the protein TatA to effect translocation. Substrates bind on the periphery of the TatBC complex, causing a reduction in the diameter of TatBC. Although the TatBC complex contains multiple copies of the signal peptide-binding TatC protomer, purified TatBC-SufI complexes contain only 1 or 2 SufI molecules according to Tarry et al., 2009. The Tat system of E. coli has been reviewed (Palmer and Berks 2012; Fröbel et al. 2012). TatBC complexes, localized to the cell poles, function with the chaparone protein, DmsD (Kostecki et al. 2010). TatA assembly and oligomerization occurs in response to substrate availability and can be reversed only by substrate transport. In contrast to TatA, the oligomeric states of TatB and TatC are not affected by substrate or the PMF, although TatB oligomerization requires TatC (Alcock et al. 2013).
A TatBC subcomplex and a homomeric TatA subcomplex comprise the TatABC complex (Fröbel et al., 2012). TatB and TatC coordinately recognize twin-arginine signal peptides and accommodate them in membrane-embedded binding pockets. Binding of the signal sequence to the Tat translocase requires the proton-motive force (PMF). When targeted in this manner, folded twin-arginine precursors induce homo-oligomerization of TatB and TatA. Ultimately, this leads to the formation of a transmembrane protein conduit that possibly consists of a pore-like TatA structure. The translocation step again is dependent on the PMF. The E. coli TatA and TatB proteins have a stable N-out C-in topology in intact cells (Koch et al., 2012). It has been proposed that the TatA transmembrane pore could self-assemble via intra- and intermolecular salt bridges (Walther et al. 2013).
Membrane protein assembly is a fundamental process in all cells. The membrane-bound Rieske iron-sulfur protein is an essential component of the cytochrome bc(1) and cytochrome b(6)f complexes, and it is exported across the energy-coupling membranes of bacteria and plants in a folded conformation in the (Tat) pathway. Although the Rieske protein in most organisms is a monotopic membrane protein, in actinobacteria, it is a polytopic protein with three TMSs. The Rieske protein of Streptomyces coelicolor requires both the Sec and the Tat pathways for assembly: the initial two TMSs integrated into the membrane in a Sec-dependent manner, whereas integration of the third TMS, and thus the correct orientation of the iron-sulfur domain, require the activity of the Tat translocase (Keller et al., 2012).
Electron microscopy displayed TatA complexes in direct contact with the membrane-stabilizing PspA protein. PspB and PspC were important for the TatA-PspA contact, but the activator protein PspF was not involved in the PspA-TatA interaction, demonstrating that basal levels of PspA already interact with TatA (Mehner et al. 2012). Elevated TatA levels caused membrane stress that induced a strictly PspBC- and PspF-dependent up-regulation of PspA. TatA complexes were found to destabilize membranes under these conditions. At native TatA levels, PspA deficiency clearly affected anaerobic TMAO respiratory growth, suggesting that energetic costs for transport of large Tat substrates such as TMAO reductase can become growth limiting in the absence of PspA (Mehner et al. 2012). The physiological role of PspA recruitment to TatA may therefore be the control of membrane stress at active translocons.
Each subunit of TatA consists of a transmembrane segment, an amphiphilic helix (APH), and a C-terminal densely charged region (DCR). The sequence of charges in the DCR is complementary to the charge pattern on the APH, suggesting that the protein can be 'zipped up' by a ladder of seven salt bridges (Walther et al. 2013). The length of the resulting hairpin matches the lipid bilayer thickness; hence a transmembrane pore could self-assemble via intra- and intermolecular salt bridges. The monomer-oligomer equilibrium of specific charge mutants was monitored (Walther et al. 2013). Similar 'charge zippers' were proposed for other membrane-associated proteins, e.g., the biofilm-inducing peptide TisB, the human antimicrobial peptide dermcidin, and the pestiviral E(RNS) protein.
Tat-mediated protein translocation initiates by signal peptide recognition and substrate binding to the TatBC complex. Upon formation of the TatBC-substrate protein complex, TatA subunits are recruited and form the protein translocation pore. TatB forms a tight complex with TatC at 1:1 molar ratio with multiple copies of both proteins. Cross-linking experiments demonstrate that TatB functions in tetrameric units and interacts with both TatC and substrate proteins. The solution structure of TatB in DPC micelles has been determined by Nuclear Magnetic Resonance (NMR) spectroscopy (Zhang et al. 2014). The structure shows an extended 'L-shape' conformation comprising four helices: a transmembrane helix (TMH) alpha1, an amphipathic helix (APH) alpha2, and two solvent exposed helices alpha3 and alpha4. The packing of TMH and APH is relatively rigid, whereas helices alpha3 and alpha4 display notably higher mobility. The observed floppiness of helices alpha3 and alpha4 allows TatB to sample a large conformational space, thus providing high structural plasticity to interact with substrate proteins of different sizes and shapes (Zhang et al. 2014).
Redox Enzyme Maturation Proteins (REMPs) are system specific chaperones, which play roles in the maturation of Tat-dependent respiratory enzymes. Kuzniatsova et al. 2016 applied the in vivo bacterial two-hybrid technique to investigate interaction of REMPs with the TatBC proteins, finding that all but the formate dehydrogenase REMP dock to TatB or TatC. They focused on the NarJ subfamily; DmsD, the REMP for dimethyl sulfoxide reductase in E. coli, had previously been shown to interact with TatB and TatC. These REMPs interact with TatC cytoplasmic loops 1, 2 and 4 with the exception of NarJ that only interacts with loops 1 and 4. An in vitro isothermal titration calorimetry study was applied to confirm the evidence of interactions between TatC fragments and DmsD chaperone. Using a peptide overlapping array, it was shown that the different NarJ subfamily REMPs interact with different regions of the TatB cytoplasmic domains. Thus, REMP chaperones play a role in targeting respiratory enzymes to the Tat system (Kuzniatsova et al. 2016).
The generalized transport reaction is:
Folded protein (cytoplasm) + energy → Folded protein (out) (periplasm of Gram-negative bacteria).