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

Abstract

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...

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

“…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.…”

Twin-arginine translocase mutations that suppress folding quality control and permit export of misfolded substrate proteins

“…Hereafter, we focused our attention on the class I suppressors. To determine whether class I suppression was related to intracellular abundance of translocase proteins, the expression level of the mutant Tat proteins was analyzed by immunoblotting (14,15). Nearly equal amounts of TatA were detected in membranes from cells expressing wt and mutant Tat translocases, whereas subtle differences in TatB expression were observed (Fig.…”

“…S3). Initial attempts to detect TatC were unsuccessful, likely because of the low expression level from pBR322, as was reported (15). To circumvent this problem, a small epitope tag was fused to the C terminus of TatC, which is known to tolerate C-terminal fusions without loss of function (15).…”

“…Using this selection, we isolated a set of suppressor mutants that rescued the export of the poorly folded α 3 B substrate; some of these could also export other misfolded proteins including α 3 A and PhoA. Although mutant translocases possessing relaxed signal peptide specificities have been reported (14,15), this work reports distinct mutant translocases that are less stringent with respect to the structural integrity of their substrates.…”

The Canonical Twin-Arginine Translocase Components Are Not Required for Secretion of Folded Green Fluorescent Protein from the Ancestral Strain of Bacillus subtilis

“…Thus, we wanted to determine if PhoD SP -GFP was secreted in a Tatdependent manner. Toward this end, we generated strains that contained individual and double deletions of the two known Tat signal peptide recognition proteins, TatCd and TatCy (24)(25)(26)71). qRT-PCR, fluorescent images of expressing cells, and Western blot analysis indicated that each Tat deletion strain expressed PhoD SP -GFP at similar levels (see Table S3 and Fig.…”