The Entire N-Terminal Half of TatC is Involved in Twin-Arginine Precursor Binding

Holzapfel, Eva; Eisner, Gottfried; Alami, Mériem; Barrett, Claire M.L.; Buchanan, Grant; Lüke, Iris; Betton, Jean‐Michel; Robinson, Colin; Palmer, Tracy; Moser, Michael J.; Müller, Matthias

doi:10.1021/bi062205b

Cited by 68 publications

(93 citation statements)

References 39 publications

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“…In agreement with these observations, recent in vitro cross-linking studies also highlighted the significance of the N terminus of TatC for signal peptide recognition. 35 As was the case with the mechanistic elucidation of the Sec export pathway, the synergism between genetic and biochemical analysis is expected to lead to deep insights that would be unlikely to arise solely by either approach.…”

Section: Discussionmentioning

confidence: 99%

Escherichia coli tatC Mutations that Suppress Defective Twin-Arginine Transporter Signal Peptides

Strauch

Georgiou

2007

Journal of Molecular Biology

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In vitro studies have suggested that the TatBC complex serves as the receptor for signal peptides targeted for export via the twin-arginine translocation (Tat) pathway. Substitution of the hallmark twin-arginine dipeptide with two lysines abrogates export of physiological substrates in all organisms. We report the isolation and characterization of suppressor mutations that allow export of an ssTor(KK)-GFP-SsrA tripartite fusion. We identified two amino acid suppressor mutations in the first cytoplasmic loop of TatC. In addition, two other amino acids in the first cytoplasmic loop exhibit epistatic suppression. Surprisingly, we also identified a suppressor mutation predicted to lie within the second periplasmic loop of TatC, a region that is not expected to interact directly with the signal peptide. The suppressor mutations allowed export of the native Esherichia coli Tat substrate trimethylamine N-oxide reductase with a twin-lysine substitution in its signal sequence. The cytoplasmic suppressor mutations conferred SDS sensitivity and partial filamentation, indicating that Tat export of authentic substrates was impaired.Keywords twin-arginine translocase; TatC; signal peptide recognition; flow cytometry; suppressorsThe bacterial twin-arginine (RR) translocation (Tat) pathway is responsible for the transport of cofactor-containing proteins and other folded polypeptides across the cytoplasmic membrane. Proteins exported via Tat contain long signal peptides featuring a conserved RR motif with a consensus sequence of S/T-R-R-x-F-L-K. 1 In Escherichia coli, the functional Tat translocon is composed of the membrane proteins TatA, TatB and TatC. Among Gram-positive bacteria, a few organisms can be found containing only two identifiable tat genes, encoding TatA and TatC homologues and lacking a tatB gene. 2-5 In Escherichia coli, TatA is purified as a series of high molecular weight homo-oligomers that form pore-like structures of varying diameters. Based on these observations, Gohlke et al. proposed that TatA forms the proteinconducting pore that assembles on demand, the size of which is determined by the dimensions of the translocated protein substrate. 6 Biochemical analyses have revealed that TatB copurifies in a complex with TatC and that this complex plays a key role in the targeting of Tat substrates to the translocon. 7-9 Based on cross-linking of in vitro translocated proteins into inverted membrane vesicles, Alami et al. discovered that TatC in particular exhibits extensive contact with the signal peptide and recognizes the RR motif. 7 * Corresponding author. E-mail address: gg@che.utexas.edu. NIH Public Access Isolation of suppressor TatC variants by fluorescence-activated cell sortingssTorA fused to the fluorescent reporter GFP-SsrA allows the facile and quantitative detection of Tat export by flow cytometry. 11 The ssTorA-GFP-SsrA (TGS) fusion is encoded by the plasmid pTGS 11 under the control of the arabinose promoter. The presence of the SsrA tag on green fluorescence protein (GFP) ensures that a...

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Section: Discussionmentioning

confidence: 99%

Escherichia coli tatC Mutations that Suppress Defective Twin-Arginine Transporter Signal Peptides

Strauch

Georgiou

2007

Journal of Molecular Biology

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“…To confirm that TatA-YFP association was the result of a productive interaction between substrate proteins and TatBC, we expressed both CueO and TatBC from plasmids in strain AyE, but this time we used a TatC variant containing the substitutions F94A and E103A that are known to block signal peptide binding (21,38). These cells failed to produce TatA-YFP assemblies, demonstrating that TatBC must be capable of binding substrate proteins for TatA association to occur (Fig.…”

Section: Coexpression With Tate Improves the Transport Activity Of Cellsmentioning

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.

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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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“…The ability of imported cpTatCs to assemble into a cpTat receptor complex with Hcf106 and to bind substrate proteins was tested in the experiment in Figure 2. As proof of concept for mutagenesis studies, this experiment also included three pea (P. sativum) cpTatC mutants based on E. coli TatC mutants impaired in substrate binding (F94A and E103A) or TatC-TatB interaction (D211A) (Buchanan et al, 2002;Holzapfel et al, 2007). The comparable cpTatC Ala substitutions are F158A, E167A, and D276A.…”

Section: Imported Cptatc Protein Assembles Into New Cptat Receptor Comentioning

confidence: 99%

“…One approach to address this combines mutagenesis with biochemical characterization and protein-protein interaction studies. This approach has been conducted to some extent with the Escherichia coli Tat system (Allen et al, 2002;Buchanan et al, 2002;Barrett et al, 2005;McDevitt et al, 2006;Holzapfel et al, 2007), but not with the plant Tat system owing to the relative difficulty of mutagenesis and gene replacement.…”

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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The Entire N-Terminal Half of TatC is Involved in Twin-Arginine Precursor Binding

Cited by 68 publications

References 39 publications

Escherichia coli tatC Mutations that Suppress Defective Twin-Arginine Transporter Signal Peptides

Escherichia coli tatC Mutations that Suppress Defective Twin-Arginine Transporter Signal Peptides

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

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