The early mature part of bacterial twin-arginine translocation (Tat) precursor proteins contributes to TatBC receptor binding

Abstract

The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial membranes. Tat precursor proteins possess a conserved twin-arginine (RR) motif in their signal peptides that is involved in the binding of the proteins to the membrane-associated TatBC receptor complex. In addition, the hydrophobic region in the Tat signal peptides also contributes to TatBC binding, but whether regions beyond the signal-peptide cleavage site are involved in this process is unknown. Here, we analyzed the contribution of the early mature protein part of the Escherichia coli trimethylamine N-oxide reductase (TorA) to productive TatBC receptor binding. We identified substitutions in the 30 amino acids immediately following the TorA signal peptide (30aa-region) that restored export of a transport-defective TorA[KQ]-30aa-MalE precursor, in which the RR residues had been replaced by a lysine-glutamine pair. Some of these substitutions increased the hydrophobicity of the N-terminal part of the 30aa-region and thereby likely enhanced hydrophobic substrate-receptor interactions within the hydrophobic TatBC substrate-binding cavity. Another class of substitutions increased the positive net charge of the region's C-terminal part, presumably leading to strengthened electrostatic interactions between the mature substrate part and the cytoplasmic TatBC regions. Furthermore, we identified substitutions in the C-terminal domains of TatB following the transmembrane segment that restored transport of various transport-defective TorA-MalE derivatives. Some of these substitutions most likely affected the orientation or conformation of the flexible, carboxy-proximal helices of TatB. Therefore, we propose that a tight accommodation of the folded mature region by TatB contributes to productive binding of Tat substrates to TatBC.

Keywords: Escherichia coli (E. coli); early mature region; membrane transport; protein export; protein folding; protein targeting; protein translocation; secretion pathway; substrate receptor; twin arginine translocation.

Figure 1.

Generation of a mutant library of TorA[KQ]-30aa-MalE by site-directed mutagenesis for the identification of suppressing mutations in the 30aa-region. The amino acid sequence of TorA[KQ]-30aa-MalE encompassing the entire TorA signal peptide (n-region, h-region, and c-region), the first 30 amino acids of the mature TorA protein (30aa-region), and the first five residues of the mature MalE protein is shown. The Tat consensus motif is underlined (dashed line); the position of the twin arginines is marked by arrows. The KQ mutation of the RR motif is highlighted in black. A total of 20 residues in the 30aa-region (Ala-40, Gln-41, Ala-42, Ala-43, Thr-44, Asp-45, Ile-48, Ser-49, Lys-50, Glu-51, Gly-52, Thr-55, Gly-56, Ser-57, His-58, Gly-60, Arg-63, Thr-65, Lys-67, and Asp-68; highlighted in black) has been separately subjected to saturation mutagenesis (*) to generate a library of TorA[KQ]-30aa-MalE reporter variants harboring single amino acid substitutions at the predetermined positions in the 30aa-region.

Figure 2.

Mutations in the 30aa-region cooperate with each other in the suppression of the export defect of a TorA[KQ]-30aa-MalE precursor protein. A, amino acid sequences of TorA-30aa-MalE reporter variants encompassing the Tat consensus motif (dashed line) in the TorA signal peptide (residues 1–39), the entire 30aa-region (residues 40–69), and the first five amino acid residues of the mature MalE protein (residues 70–439). Positions of the particular mutations in the 30aa-region are highlighted in red and underlined. The KQ mutation of the RR motif is highlighted in black. B, export of the various TorA[KQ]-30aa-MalE reporter variants has been analyzed by MMM/MCM plate assays in the presence (Tat+) or absence (Tat−) of the Tat translocase in E. coli GSJ101 and on the protein level. The phenotypes of the respective strains on MMM (−, no growth; +, slow growth; ++, moderate growth; +++, good growth) and MCM (pale, light red, pink, or red color of colonies) after 24 h of incubation are shown in the boxes at the bottom of the figure. Cells were fractionated into a periplasmic fraction and a combined cytosol/membrane fraction by EDTA-lysozyme spheroplasting. The samples of the fractions corresponding to an identical amount of cells were subjected to SDS-PAGE and immunoblotting using anti-MalE antibodies. Subsequently, relative export efficiencies were calculated by determining the amount of exported MalE protein in the periplasmic fraction of strains GSJ101 coexpressing the tatABCE genes and TorA-30aa-MalE or one of the mutated TorA[KQ]-30aa-MalE reporter variants in at least three different independent experiments via quantification of the chemiluminescence signals. The signals were recorded by a CCD camera and subsequently analyzed by the program AIDA 4.50 (Raytest). The average values are indicated by horizontal marker lines and standard deviations by error bars. The relative export efficiency of the positive control GSJ101 (pTorA-30aa-MalE and pHSG-TatABCE) was set to 100% (wt; lane 1). The other samples correspond to GSJ101 coexpressing the tatABCE genes and the export-defective TorA[KQ]-30aa-MalE reporter (KQ; lane 2) or one of the TorA[KQ]-30aa-MalE reporter variants containing the single, double, or triple mutations in the 30aa-region indicated below lanes 3–14. wt, WT.

Figure 3.

Mutations in the h-region of the TorA signal peptide and the 30aa-region cooperate with each other in the suppression of the export defect of a TorA[KQ]-30aa-MalE precursor protein. A, amino acid sequences of TorA-30aa-MalE reporter variants encompassing the Tat consensus motif (dashed line) and the entire h-region of the TorA signal peptide (residues 1–39), the entire 30aa-region (residues 40–69), and the first five amino acid residues of the mature MalE protein (residues 70–439). Positions of the particular mutations in the h-region of the TorA signal peptide (or, in case of the mutation A16V, at the boundary between the n-region with the Tat consensus motif and the h-region) and/or the 30aa-region are highlighted in red and underlined. The KQ mutation of the RR motif is highlighted in black. B, export of the various TorA[KQ]-30aa-MalE reporter variants has been analyzed by MMM/MCM plate assays in the presence (Tat+) or absence (Tat−) of the Tat translocase in E. coli GSJ101 and on the protein level. The phenotypes of the respective strains on MMM (−, no growth; +, slow growth; ++, moderate growth; +++, good growth) and MCM (pale, light red, pink, or red color of colonies) after 24 h of incubation are shown in the boxes at the bottom of the figure. Cells were fractionated into a periplasmic fraction and a combined cytosol/membrane fraction by EDTA-lysozyme spheroplasting. The samples of the fractions corresponding to an identical amount of cells were subjected to SDS-PAGE and immunoblotting using anti-MalE antibodies. Subsequently, relative export efficiencies were calculated by determining the amount of exported MalE protein in the periplasmic fraction of strains GSJ101 coexpressing the tatABCE genes and TorA-30aa-MalE or one of the mutated TorA[KQ]-30aa-MalE reporter variants in at least three different independent experiments via quantification of the chemiluminescence signals. The signals were recorded by a CCD camera and subsequently analyzed by the program AIDA 4.50 (Raytest). The average values are indicated by horizontal marker lines and standard deviations by error bars. The relative export efficiency of the positive control GSJ101 (pTorA-30aa-MalE, pHSG-TatABCE) was set to 100% (wt; lane 1). The other samples correspond to GSJ101 coexpressing the tatABCE genes and the export-defective TorA[KQ]-30aa-MalE reporter (KQ; lane 2) or one of the TorA[KQ]-30aa-MalE reporter variants containing the single, double, or triple mutations in the h-region of the TorA signal peptide and/or the 30aa-region indicated below the lanes 3–18. wt, WT.

Figure 4.

Combinations of mutations in the 30aa-region and the mutation L9F in TatC synergistically suppress the export-defect of a TorA[KQ]-30aa-MalE precursor protein. A–D, subcellular localization of TorA-30aa-MalE-derived polypeptides. Cells were fractionated into a periplasmic (P) fraction and a combined cytosol/membrane (C/M) fraction by EDTA-lysozyme spheroplasting. The samples of the fractions corresponding to an identical amount of cells were subjected to SDS-PAGE and immunoblotting using anti-MalE antibodies. The positive controls were E. coli GSJ101 containing plasmids pTorA-30aa-MalE and pHSG-TatABCE (lane 1) or pHSG-TatABC[L9F]E (lane 2). The other samples correspond to GSJ101 coexpressing the WT tatABCE or mutated tatABC[L9F]E genes and the export-defective TorA[KQ]-30aa-MalE reporter (lanes 3 and 4, respectively) or one of the TorA[KQ]-30aa-MalE reporter variants containing the single mutations or the double mutations in the 30aa-region as indicated above the lanes (lanes 5–20). wt, WT; p, WT or mutated TorA-30aa-MalE precursor in the C/M fraction; m, mature form of MalE in the P fraction; asterisk, WT or mutated TorA-30aa-MalE-derived degradation products in the C/M fraction. Positions of molecular weight markers are indicated on the left margin. The phenotypes of the respective strains on MMM (−, no growth; +, slow growth; ++, moderate growth; +++, good growth) and MCM (pale, light red (light r.), pink, red) agar plates after 24 h of incubation are shown in the boxes at the bottom of the figure. E, relative export efficiencies of the analyzed TorA-30aa-MalE reporter variants in strains expressing the WT or mutant TatABC[L9F]E translocase. The amount of exported MalE protein in the P fraction of strains GSJ101 coexpressing the WT tatABCE or mutated tatABC[L9F]E genes and TorA-30aa-MalE, TorA[KQ]-30aa-MalE, or one of the mutated TorA[KQ]-30aa-MalE reporter variants was determined in at least three different independent experiments via quantification of the chemiluminescence signals. The signals were recorded by a CCD camera and subsequently analyzed by the program AIDA 4.50 (Raytest). The average values are indicated by horizontal marker lines; standard deviations by error bars. The relative export efficiency of the positive control GSJ101 (pTorA-30aa-MalE, pHSG-TatABCE) was set to 100%. The lane numbers in E correspond to the lane numbers in A–D.

Figure 5.

Model of possible TatBC–precursor interactions. The framed light gray bar represents the lipid bilayer. For clarity, only one TatB monomer (checkered cylinder) and two adjacent TatC monomers (diagonally hatched cylinders) of the multimeric TatBC receptor complex are shown. Each TatC monomer is depicted by six transmembrane helices. The TatB monomer consists of a membrane-embedded transmembrane helix (TMH), a cytosolic amphipathic helix (APH), and flexible C-terminal domains (helices α3 and α4) encapsulating the folded Tat substrate. The membrane-embedded TorA signal peptide with the h-region forming an α-helix (black cylinder) and the following 30aa-region of the TorA-30aa-MalE precursor protein is represented by a black line; the folded MalE mature region is represented by a black ellipse. The positions of selected suppressor mutations in the membrane-embedded N-terminal half of the 30aa-region are indicated by red dots, substitutions in the cytoplasmically-located C-terminal half of the 30aa-region by blue dots, and the TatB-located suppressing mutations of the TorA[KQ]-30aa-MalE export defect by green dots. The indicated positions of the newly introduced hydrophobic (red arrows) or electrostatic (blue arrows) precursor–TatBC interactions by the 30aa-region-located mutations are speculative. The effects of the more C-terminal TatB mutations (T51A, T51I, T75M, P76L, and A89V) on the orientation/conformation of the flexible cytosolic domains are likewise hypothetical. A possible scenario could be that, upon insertion of the signal peptide and the partially unfolded N-terminal part of the early mature protein into the hydrophobic TatBC-binding cavity, these substitutions alter the conformation or orientation of the C-terminal domains of TatB such that they allow them to wrap more tightly around the folded part of the non-native substrate MalE, thereby promoting a stronger fixation (“trapping”) of the otherwise too weakly bound Tat precursor to the Tat translocase.