Mapping the Signal Peptide Binding and Oligomer Contact Sites of the Core Subunit of the Pea Twin Arginine Protein Translocase

Ma, Xianyue; Cline, Kenneth

doi:10.1105/tpc.112.107409

Cited by 37 publications

(75 citation statements)

References 55 publications

(105 reference statements)

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“…This finding is consistent with previous observations that these two proteins copurify in detergent solution as a large TatBC complex and that the size and composition of the complex are not significantly altered by substrate binding (19,22,45,49,50). It is also consistent with the observation that TatBC complexes do not exhibit the exchange of TatC subunits that would be expected if TatC undergoes assembly cycles (23). We find that the TatB protein has a lower oligomer state if TatC is absent but that TatC oligomerization is independent of TatB.…”

Section: Discussionsupporting

confidence: 82%

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Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system

Alcock

Baker

Greene

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

170

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The twin-arginine translocation (Tat) machinery transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. It has been inferred that the Tat translocation site is assembled on demand by substrate-induced association of the protein TatA. We tested this model by imaging YFP-tagged TatA expressed at native levels in living Escherichia coli cells in the presence of low levels of the TatA paralogue TatE. Under these conditions the TatA-YFP fusion supports full physiological Tat transport activity. In agreement with the TatA association model, raising the number of transport-competent substrate proteins within the cell leads to an increase in the number of large TatA complexes present. Formation of these complexes requires both a functional TatBC substrate receptor and the transmembrane proton motive force (PMF). Removing the PMF causes TatA complexes to dissociate, except in strains with impaired Tat transport activity. Based on these observations we propose that TatA assembly reaches a critical point at which oligomerization can be reversed only by substrate transport. In contrast to TatA-YFP, the oligomeric states of TatB-YFP and TatC-YFP fusions are not affected by substrate or the PMF, although TatB-YFP oligomerization does require TatC.

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Section: Discussionsupporting

confidence: 82%

“…Tat-signal peptides are recognized at the membrane by a TatBC receptor complex (18)(19)(20)(21)(22)(23). Substrate protein binding to TatBC causes a PMF-dependent recruitment of TatA (and presumably TatE) to assemble the active translocation site (18,24).…”

mentioning

confidence: 99%

Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system

Alcock

Baker

Greene

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

170

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“…3A shows residues of chloroplast TatC (colored orange and red) that, when mutated to alanine, impair signal peptide binding. Residues colored red (glutamate) are thought to coordinate the arginine guanidinium groups; residues colored green direct disulfide cross-linking to RR proximal residues of the signal peptide (56). Complementary evidence also suggests that signal peptide H domain uniformly binds to the TatB TM, which is simultaneously associated with TatC TM5.…”

Section: Substratementioning

confidence: 91%

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

Cline

2015

Journal of Biological Chemistry

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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...

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“…The TatB and TatC proteins form a multivalent complex that binds Tat substrates through their twin arginine signal peptides (18)(19)(20). Numerous experiments have shown that the TatC component recognizes the twin arginine motif (21)(22)(23)(24)(25)(26), whereas TatB is close to the signal peptide h-region (27,28). Signal peptides have been shown to penetrate deeply into the TatBC complex (29) and, in thylakoids at least, this deep-binding mode may be modulated by the transmembrane proton electrochemical gradient (PMF) (30).…”

mentioning

confidence: 99%

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

Huang

Alcock

Kneuper

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The twin-arginine protein translocation (Tat) system mediates transport of folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. The Tat system of Escherichia coli is made up of TatA, TatB, and TatC components. TatBC comprise the substrate receptor complex, and active Tat translocases are formed by the substrate-induced association of TatA oligomers with this receptor. Proteins are targeted to TatBC by signal peptides containing an essential pair of arginine residues. We isolated substitutions, locating to the transmembrane helix of TatB that restored transport activity to Tat signal peptides with inactivating twin arginine substitutions. A subset of these variants also suppressed inactivating substitutions in the signal peptide binding site on TatC. The suppressors did not function by restoring detectable signal peptide binding to the TatBC complex. Instead, site-specific cross-linking experiments indicate that the suppressor substitutions induce conformational change in the complex and movement of the TatB subunit. The TatB F13Y substitution was associated with the strongest suppressing activity, even allowing transport of a Tat substrate lacking a signal peptide. In vivo analysis using a TatA-YFP fusion showed that the TatB F13Y substitution resulted in signal peptide-independent assembly of the Tat translocase. We conclude that Tat signal peptides play roles in substrate targeting and in triggering assembly of the active translocase.protein transport | twin arginine signal peptide | Tat pathway | genetic suppressor

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Mapping the Signal Peptide Binding and Oligomer Contact Sites of the Core Subunit of the Pea Twin Arginine Protein Translocase

Cited by 37 publications

References 55 publications

Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system

Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system

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

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

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