Escherichia coli Twin Arginine (Tat) Mutant Translocases Possessing Relaxed Signal Peptide Recognition Specificities

Kreutzenbeck, Peter; Kröger, Carsten; Lausberg, Frank; Blaudeck, Natascha; Sprenger, Georg A.; Freudl, Roland

doi:10.1074/jbc.m610126200

Cited by 65 publications

(125 citation statements)

References 33 publications

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“…A first, superficial targeting step would not exclusively depend on an intact RR pair, if it involved recognition of an extended sequence area within an RR signal peptide. Along this line, the recent isolation of distinct suppressor mutants in tatB and tatC that tolerate a KQ signal peptide and retain the ability to accept the RR wild type (17) suggests a large binding pocket with multiple contact points. Furthermore, less strictly conserved residues of the SRRxFLK consensus sequence likely contribute to the specificity of targeting (48,49).…”

Section: Discussionmentioning

confidence: 99%

“…We would predict that this recognition occurs deeper in the membrane. Recent findings indicate that an RR signal peptide must be able to interact with TatBC also within the plane of the lipid bilayer: some suppressor mutations of a KQ variant of TorA-MalE map to the N terminus of TatB predicted to lie on the trans-side of the membrane (17) as well as to a predicted periplasmic loop of TatC (18). Substitution of Phe for Val two residues downstream of the RR pair of the thylakoid precursor tOE17 results in a protease-resistant insertion of the signal sequence into the thylakoid Hcf106-cpTatC receptor (50), which together with other results prompted these authors to suggest a similar two-stage binding model.…”

Section: Discussionmentioning

confidence: 99%

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Following the Path of a Twin-arginine Precursor along the TatABC Translocase of Escherichia coli

Panahandeh

Maurer

Moser

et al. 2008

Journal of Biological Chemistry

104

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The twin-arginine translocation (Tat) machinery present in bacterial and thylakoidal membranes is able to transport fully folded proteins. Consistent with previous in vivo data, we show that the model Tat substrate TorA-PhoA is translocated by the TatABC translocase of Escherichia coli inner membrane vesicles, only if the PhoA moiety was allowed to fold by disulfide bond formation. Although even unfolded TorA-PhoA was found to physically associate with the Tat translocase of the vesicles, site-specific cross-linking revealed a perturbed interaction of the signal sequence of unfolded TorA-PhoA with the TatBC receptor site. Some of the folded TorA-PhoA precursor accumulated in a partially protease-protected membrane environment, from where it could be translocated into the lumen of the vesicles upon re-installation of an H ؉ -gradient. Translocation arrest occurred in immediate vicinity to TatA. Consistent with a neighborhood to TatA, TorA-PhoA remained protease-resistant in the presence of detergents that are known to preserve the oligomeric structures of TatA. Moreover, entry of TorA-PhoA to the protease-protected environment strictly required the presence of TatA. Collectively, our results are consistent with some degree of quality control by TatBC and a recruitment of TatA to a folded substrate that has functionally engaged the twin-arginine translocase.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Following the Path of a Twin-arginine Precursor along the TatABC Translocase of Escherichia coli

Panahandeh

Maurer

Moser

et al. 2008

Journal of Biological Chemistry

104

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“…However, in a search for tatC mutants that allow export of a defective ssTorA(KQ)-MBP fusion, Kreutzenbeck et al reported the isolation of the amino acid substitution E8K located at the N terminus of TatB, a domain that is predicted to be localized in the periplasm. 12 The finding that mutations in periplasmic residues in TatC or in TatB can impact the recognition of the signal sequence highlights the complexity of the organization of the Tat translocon. As has been noted by Kreutzenbeck et al, mutations in residues exposed to the periplasmic side could result in a reorganization of the tertiary structure of TatC or TatB.…”

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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“…To test if the Tat-like targeting signal is functional we analyzed the import and assembly of a mutated Rip protein. We mutated the potential Arabidopsis Rip Tat signal of KR to QQ; glutamines were chosen because substitutions with glutamines had previously been shown to completely abolish Tat pathway export in bacteria (Kreutzenbeck et al, 2007). Using in vitro import assays into isolated Arabidopsis mitochondria it was observed that Rip-KR imports and assembles into complex III and the super-complex of I and III very efficiently, as seen by the increase in radioactive signal over time (Fig.…”

Section: The Arabidopsis Rip Protein Requires a Tat Signal And A δPh mentioning

confidence: 99%

Plant mitochondria contain the protein translocase subunits TatB and TatC

Carrie

Weißenberger

Soll

2016

Journal of Cell Science

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Twin-arginine translocation (Tat) pathways have been wellcharacterized in bacteria and chloroplasts. Genes encoding a TatC protein are found in almost all plant mitochondrial genomes but to date these have not been extensively investigated. For the first time it could be demonstrated that this mitochondrial-encoded TatC is a functional gene that is translated into a protein in the model plant Arabidopsis thaliana. A TatB-like subunit localized to the inner membrane was also identified that is nuclear-encoded and is essential for plant growth and development, indicating that plants potentially require a Tat pathway for mitochondrial biogenesis.

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Escherichia coli Twin Arginine (Tat) Mutant Translocases Possessing Relaxed Signal Peptide Recognition Specificities

Cited by 65 publications

References 33 publications

Following the Path of a Twin-arginine Precursor along the TatABC Translocase of Escherichia coli

Following the Path of a Twin-arginine Precursor along the TatABC Translocase of Escherichia coli

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

Plant mitochondria contain the protein translocase subunits TatB and TatC

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