Mechanistic Aspects of Folded Protein Transport by the Twin Arginine Translocase (Tat)

Abstract

The twin arginine translocase (Tat) transports folded proteins of widely varying size across ionically tight membranes with only 2-3 components of machinery and the proton motive force. Tat operates by a cycle in which the receptor complex combines with the pore-forming component to assemble a new translocase for each substrate. Recent data on component and substrate organization in the receptor complex and on the structure of the pore complex inform models for translocase assembly and translocation. A translocation mechanism involving local transient bilayer rupture is discussed.Cells and membranes contain multiple protein translocases, i.e. the enzymes that catalyze protein translocation across or into membranes. This allows for specific targeting to distinct cellular locations and, through varied transport mechanisms, solutions to a range of translocation problems. For example, the prokaryotic and endoplasmic reticulum Sec system and several other translocases transport proteins in a mostly unfolded conformation. This permits a single mechanistic strategy for those substrates that can be unfolded for translocation and folded following transport. By contrast, the twin arginine translocase (Tat) 2 system transports proteins that are in a folded conformation during translocation. The Tat system is present in most bacteria, in some archaea, in chloroplasts of plants and algae, and in some mitochondria (1-5). The prevalence of Tat in prokaryotes and prokaryote-derived organelles argues that Tat is an ancient translocation system. The fact that Tat operates with as few as two membrane components of machinery (TatA and TatC) (6) and the proton motive force (PMF) implies a simple mechanism. Although it may be mechanistically simple, Tat behaves in mystifying ways and performs astonishing feats. It exists as oligomeric structures, with a multivalent receptor complex composed of TatC and TatB multimers and a "pore" complex of TatA oligomers that are thought to form the protein-conducting element. Tat can transport folded proteins from ϳ20 Å (7) to ϳ70 Å (8). It can transport heterodimers, where one subunit has the signal peptide and the other hitchhikes the ride (9), and cross-linked tetramers, where each subunit is bound via its own signal peptide (10). Tat will even transport engineered unstructured polypeptides (11,12). Thus, Tat has solved the daunting mechanistic challenge of transporting both large and small protein structures without opening large holes that would dissipate the PMF.The Tat substrate repertoire depends on the organism and ranges from 1 to ϳ150 substrates (reviewed in Ref. 2). Tat plays critical roles in respiratory and photosynthetic energy production, animal and plant pathogenesis, symbiosis, etc. (reviewed in Refs. 2,8,[13][14][15]. Among reasons that proteins require transport in a folded conformation include: the absence of folding and cofactor insertion machinery on the trans side of the membrane, the need to control metal cofactor specificity, and the fact that some proteins rapidly fold up...

TatE as a Regular Constituent of Bacterial Twin-arginine Protein Translocases

“…Because we found TatE cross-linked to TatC, it might also affect the way in which TatA and/or TatB associate with TatC. Common binding sites of TatA and TatB on TatC have recently been demonstrated, and it has been suggested that TatB might control the access and oligomerization of TatA on the inside of concentric TatBC assemblies (6,15,17). As we have shown here, TatE, at high concentrations, seems to be able to displace TatA from TatC.…”

“…In cells overexpressing TatE-GFP, the major TatE-immunoreactive material had the expected molecular mass of the TatE-GFP fusion (approximately 37 kDa; Fig. 2A, lanes [3][4][5][6]. Expression of TatE-GFP did not noticeably influence the amount of the 9-kDa TatE band, demonstrating stability of the TatE-GFP construct.…”

“…[1][2][3][4][5][6]. Whereas TatC is a hexahelical membrane protein (7,8), TatA, TatB, and TatE consist of only one transmembrane helix, followed by an amphipathic helix and an unstructured C terminus on the cis side of the membrane.…”

The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding

“…TatA is a key component of the Tat system and considered to locally weaken the membrane, thereby -in conjunction with the other Tat system components -allowing the passage of folded proteins (19,18). TatA is anchored by its Nterminus in the cytoplasmic membrane (20).…”

A signal sequence suppressor mutant that stabilizes an assembled state of the twin arginine translocase

“…The twin-arginine protein translocation (Tat) pathway operates in parallel to the Sec pathway to transport folded proteins across the prokaryotic cytoplasmic membrane and the thylakoid membrane of plant chloroplasts (10,11). Proteins are targeted to the Tat pathway by N-terminal signal peptides that contain a conserved twin arginine motif at the n-region/h-region boundary (12,13) (Fig.…”