Stoichiometry for binding and transport by the twin arginine translocation system

Celedon, Jose M.; Cline, Kenneth

doi:10.1083/jcb.201201096

Cited by 47 publications

(79 citation statements)

References 53 publications

(89 reference statements)

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“…8). Similar considerations apply if multiple substrate-binding sites within the TatBC complex are able to access the same TatA assembly (20,48).…”

Section: Discussionmentioning

confidence: 99%

“…However, during transport at high substrate concentrations, binding of the next substrate molecule to the TatBC complex might occur before TatA dissociation has started or before it has been fully completed. Under these conditions it is implicit in our model that TatA sites can be reused, as recently suggested (20), or regenerated from a partially disassembled state by reversing the direction of the disassembly equilibrium upon substrate binding (Fig. 8).…”

Section: Discussionmentioning

confidence: 99%

“…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%

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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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“…8). Similar considerations apply if multiple substrate-binding sites within the TatBC complex are able to access the same TatA assembly (20,48).…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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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show abstract

“…Binding of the signal peptide triggers assembly of Tha4 (Mori and Cline, 2002). In nontransporting membranes, Tha4 appears to be a homotetramer, whereas in the translocase, it assembles as homooligomers of ;20 to 25 Tha4 protomers (Leake et al, 2008;Dabney-Smith and Cline, 2009;Celedon and Cline, 2012). For this and other reasons, Tha4 (TatA) is thought to form the protein-conducting structure.…”

Section: Introductionmentioning

confidence: 99%

“…cpTatC (TatC) is the primary receptor for the signal peptide (Alami et al, 2003;Gérard and Cline, 2006), although Hcf106 (TatB) also contacts the signal peptide to a lesser extent. Under saturating conditions, each cpTatC within a receptor complex binds a substrate protein (Celedon and Cline, 2012). Kinetic analysis of transport showed that each cpTatC-Hcf106 pair independently transports its bound substrate in an apparently stochastic manner (Celedon and Cline, 2012).…”

Section: Introductionmentioning

confidence: 99%

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

Cline

2013

The Plant Cell

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Twin arginine translocation (Tat) systems of thylakoid and bacterial membranes transport folded proteins using the proton gradient as the sole energy source. Tat substrates have hydrophobic signal peptides with an essential twin arginine (RR) recognition motif. The multispanning cpTatC plays a central role in Tat operation: It binds the signal peptide, directs translocase assembly, and may facilitate translocation. An in vitro assay with pea (Pisum sativum) chloroplasts was developed to conduct mutagenesis and analysis of cpTatC functions. Ala scanning mutagenesis identified mutants defective in substrate binding and receptor complex assembly. Mutations in the N terminus (S1) and first stromal loop (S2) caused specific defects in signal peptide recognition. Cys matching between substrate and imported cpTatC confirmed that S1 and S2 directly and specifically bind the RR proximal region of the signal peptide. Mutations in four lumen-proximal regions of cpTatC were defective in receptor complex assembly. Copurification and Cys matching analyses suggest that several of the lumen proximal regions may be important for cpTatC-cpTatC interactions. Surprisingly, RR binding domains of adjacent cpTatCs directed strong cpTatC-cpTatC cross-linking. This suggests clustering of binding sites on the multivalent receptor complex and explains the ability of Tat to transport cross-linked multimers. Transport of substrate proteins cross-linked to the signal peptide binding site tentatively identified mutants impaired in the translocation step.

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Thylakoid‐integrated recombinant Hcf106 participates in the chloroplast twin arginine transport system

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New

et al. 2018

Plant Direct

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The chloroplast twin arginine transport (cpTat) system distinguishes itself as a protein transport pathway by translocating fully folded proteins, using the proton‐motive force (PMF) as the sole source of energy. The cpTat pathway is evolutionarily conserved with the Tat pathway found in the plasma membrane of many prokaryotes. The cpTat (Escherichia coli) system uses three proteins, Tha4 (TatA), Hcf106 (TatB), and cpTatC (TatC), to form a transient translocase allowing the passage of precursor proteins. Briefly, cpTatC and Hcf106, with Tha4, form the initial receptor complex responsible for precursor protein recognition and binding in an energy‐independent manner, while a separate pool of Tha4 assembles with the precursor‐bound receptor complex in the presence the PMF. Analysis by blue‐native polyacrylamide gel electrophoresis (BN‐PAGE) shows that the receptor complex, in the absence of precursor, migrates near 700 kDa and contains cpTatC and Hcf106 with little Tha4 remaining after detergent solubilization. To investigate the role that Hcf106 may play in receptor complex oligomerization and/or stability, systematic cysteine substitutions were made in positions from the N‐terminal transmembrane domain to the end of the predicted amphipathic helix of the protein. BN‐PAGE analysis allowed us to identify the locations of amino acids in Hcf106 that were critical for interacting with cpTatC. Oxidative cross‐linking allowed us to map interactions of the transmembrane domain and amphipathic helix region of Hcf106. In addition, we showed that in vitro expressed, integrated Hcf106 can interact with the precursor signal peptide domain and imported cpTatC, strongly suggesting that a subpopulation of the integrated Hcf106 is participating in competent cpTat complexes.

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Stoichiometry for binding and transport by the twin arginine translocation system

Abstract: Stoichiometry determined in this study suggests that the fully loaded and assembled Tat translocase is an ∼2.2-megadalton complex that can individually transport eight precursor proteins or cooperatively transport multimeric precursors.

Cited by 47 publications

References 53 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

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

Thylakoid‐integrated recombinant Hcf106 participates in the chloroplast twin arginine transport system

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