Transport of Folded Proteins by the Tat System.

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.

Routing of thylakoid lumen proteins by the chloroplast twin arginine transport pathway.

Thylakoids are complex sub-organellar membrane systems whose role in photosynthesis makes them critical to life. Thylakoids require the coordinated expression of both nuclear- and plastid-encoded proteins to allow rapid response to changing environmental conditions. Transport of cytoplasmically synthesized proteins to thylakoids or the thylakoid lumen is complex; the process involves transport across up to three membrane systems with routing through three aqueous compartments. Protein transport in thylakoids is accomplished by conserved ancestral prokaryotic plasma membrane translocases containing novel adaptations for the sub-organellar location. This review focuses on the evolutionarily conserved chloroplast twin arginine transport (cpTat) pathway. An overview is provided of known aspects of the cpTat components, energy requirements, and mechanisms with a focus on recent discoveries. Some of the most exciting new studies have been in determining the structural architecture of the membrane complex involved in forming the point of passage for the precursor and binding features of the translocase components. The cpTat system is of particular interest because it transports folded protein domains using only the proton motive force for energy. The implications for mechanism of translocation by recent studies focusing on interactions between membrane Tat components and with the translocating precursor will be discussed.

Structure and dynamics of plant TatA in micelles and lipid bilayers studied by solution NMR.

The twin-arginine translocase (Tat) transports folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. In Gram-negative bacteria and chloroplasts, the translocon consists of three subunits, TatA, TatB, and TatC, of which TatA is responsible for the actual membrane translocation of the substrate. Herein we report on the structure, dynamics, and lipid interactions of a fully functional C-terminally truncated 'core TatA' from Arabidopsis thaliana using solution-state NMR. Our results show that TatA consists of a short N-terminal transmembrane helix (TMH), a short connecting linker (hinge) and a long region with propensity to form an amphiphilic helix (APH). The dynamics of TatA were characterized using 15 N relaxation NMR in combination with model-free analysis. The TMH has order parameters characteristic of a well-structured helix, the hinge is somewhat less rigid, while the APH has lower order parameters indicating structural flexibility. The TMH is short with a surprisingly low protection from solvent, and only the first part of the APH is protected to some extent. In order to uncover possible differences in TatA's structure and dynamics in detergent compared to in a lipid bilayer, fast-tumbling bicelles and large unilamellar vesicles were used. Results indicate that the helicity of TatA increases in both the TMH and APH in the presence of lipids, and that the N-terminal part of the TMH is significantly more rigid. The results indicate that plant TatA has a significant structural plasticity and a capability to adapt to local environments.

In silico analysis and experimental validation of lipoprotein and novel Tat signal peptides processing in Anabaena sp. PCC7120.

Signal peptide (SP) plays a pivotal role in protein translocation. Lipoprotein- and twin arginine translocase (Tat) dependent signal peptides were studied in All3087, a homolog of competence protein of Synechocystis PCC6803 and in two putative alkaline phosphatases (ALPs, Alr2234 and Alr4976), respectively. In silico analysis of All3087 is shown to possess the characteristics feature of competence proteins such as helix-hairpin-helix, N and C-terminal HKD endonuclease domain, calcium binding domain and N-terminal lipoprotein signal peptide. The SP recognition-cleavage site in All3087 was predicted (AIA-AC) using SignalP while further in-depth analysis using Pred-Lipo and WebLogo analysis for consensus sequence showed it as IAA-C. Activities of putative ALPs were confirmed by heterologous overexpression, activity assessment and zymogram analysis. ALP activity in Anabaena remains cell bound in log-phase, but during late log/stationary phase, an enhanced ALP activity was detected in extracellular milieu. The enhancement of ALP activity during stationary phase was not only due to inorganic phosphate limitation but also contributed by the presence of novel bipartite Tat-SP. The Tat signal transported the folded active ALPs to the membrane, followed by anchoring into the membrane and successive cleavage enabling transportation of the ALPs to the extracellular milieu, because of bipartite architecture and processing of transit Tat-SP.

Influence of GTP on system specific chaperone - Twin arginine signal peptide interaction.

Many bacterial respiratory redox enzymes depend on the twin-arginine translocase (Tat) system for translocation and membrane insertion. Tat substrates contain an N-terminal twin-arginine (SRRxFLK) motif serving as the targeting signal towards the translocon. Many Tat substrates have a system specific chaperone - redox enzyme maturation protein (REMP) - for final folding and assembly prior to Tat binding. The REMP DmsD strongly interacts with the twin-arginine motif of the DmsA signal sequence of dimethyl sulfoxide (DMSO) reductase. In this study, we have utilized the in vitro protein-protein interaction technique of an affinity pull down assay, as well as protein thermal stability measurement via differential scanning fluorimetry (DSF) to investigate the interaction of guanosine nucleotides (GNPs) with DmsD. Here we have shown highly cooperative binding of DmsD with GTP. A dissociative ligand-binding style isotherm was generated upon GTP titration into the DmsD:DmsAL interaction, yielding sigmoidal release of DmsD with a Hill coefficient of 2.09 and a dissociation constant of 0.99 mM. DSF further illustrated the change in thermal stability upon DmsD interaction with DmsAL and GTP. These results imply the possibility of DmsD detection and binding of GTP during the DMSO protein maturation mechanism, from ribosomal translation to membrane targeting and final assembly. Conceivably, GTP is shown to act as a molecular regulator in the biochemical pathway.

Initial assembly steps of a translocase for folded proteins.

The so-called Tat (twin-arginine translocation) system transports completely folded proteins across cellular membranes of archaea, prokaryotes and plant chloroplasts. Tat-directed proteins are distinguished by a conserved twin-arginine (RR-) motif in their signal sequences. Many Tat systems are based on the membrane proteins TatA, TatB and TatC, of which TatB and TatC are known to cooperate in binding RR-signal peptides and to form higher-order oligomeric structures. We have now elucidated the fine architecture of TatBC oligomers assembled to form closed intramembrane substrate-binding cavities. The identification of distinct homonymous and heteronymous contacts between TatB and TatC suggest that TatB monomers coalesce into dome-like TatB structures that are surrounded by outer rings of TatC monomers. We also show that these TatBC complexes are approached by TatA protomers through their N-termini, which thereby establish contacts with TatB and membrane-inserted RR-precursors.

The twin-arginine protein translocation pathway.

The twin-arginine translocation (Tat) system, found in prokaryotes, chloroplasts, and some mitochondria, allows folded proteins to be moved across membranes. How this transport is achieved without significant ion leakage is an intriguing mechanistic question. Tat transport is mediated by complexes formed from small integral membrane proteins from just two protein families. Atomic-resolution structures have recently been determined for representatives of both these protein families, providing the first molecular-level glimpse of the Tat machinery. I review our current understanding of the mechanism of Tat transport in light of these new structural data.