Limits...
Genetic evidence for a tight cooperation of TatB and TatC during productive recognition of twin-arginine (Tat) signal peptides in Escherichia coli.

Lausberg F, Fleckenstein S, Kreutzenbeck P, Fröbel J, Rose P, Müller M, Freudl R - PLoS ONE (2012)

Bottom Line: Mutations were identified in the extreme amino-terminal regions of TatB and TatC that synergistically suppressed the export defect of TorA(D(+2))-MalE when present in pairwise or triple combinations.The observed synergistic suppression activities were even more pronounced in the restoration of membrane translocation of another export-defective precursor, TorA(KQ)-MalE, in which the conserved twin arginine residues had been replaced by lysine-glutamine.Collectively, these findings indicate that the extreme amino-terminal regions of TatB and TatC cooperate tightly during recognition and productive binding of Tat-dependent precursor proteins and, furthermore, that TatB and TatC are both involved in the formation of a specific signal peptide binding site that reaches out as far as the end of the TatB transmembrane segment.

View Article: PubMed Central - PubMed

Affiliation: Institut für Bio- und Geowissenschaften 1, Biotechnologie, Forschungszentrum Jülich GmbH, Jülich, Germany.

ABSTRACT
The twin arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane of bacteria. Tat signal peptides contain a consensus motif (S/T-R-R-X-F-L-K) that is thought to play a crucial role in substrate recognition by the Tat translocase. Replacement of the phenylalanine at the +2 consensus position in the signal peptide of a Tat-specific reporter protein (TorA-MalE) by aspartate blocked export of the corresponding TorA(D(+2))-MalE precursor, indicating that this mutation prevents a productive binding of the TorA(D(+2)) signal peptide to the Tat translocase. Mutations were identified in the extreme amino-terminal regions of TatB and TatC that synergistically suppressed the export defect of TorA(D(+2))-MalE when present in pairwise or triple combinations. The observed synergistic suppression activities were even more pronounced in the restoration of membrane translocation of another export-defective precursor, TorA(KQ)-MalE, in which the conserved twin arginine residues had been replaced by lysine-glutamine. Collectively, these findings indicate that the extreme amino-terminal regions of TatB and TatC cooperate tightly during recognition and productive binding of Tat-dependent precursor proteins and, furthermore, that TatB and TatC are both involved in the formation of a specific signal peptide binding site that reaches out as far as the end of the TatB transmembrane segment.

Show MeSH

Related in: MedlinePlus

Effect of mutations at the +2 position in the Tat consensus motif.Cells were fractionated into a periplasmic (P) and a combined cytosol/membrane fraction (C/M) by EDTA-lysozyme spheroplasting. The samples were subjected to SDS-PAGE and immunoblotting using anti-MalE antibodies. The positive control was E. coli GSJ101 containing plasmids pTorA-MalE and pHSG-TatABCE (lane 1). The other samples correspond to GSJ101 containing plasmid pHSG-TatABCE in addition to pTorA(S+2)-MalE (lane 2), pTorA(R+2)-MalE (lane 3), or pTorA(D+2)-MalE (lane 4). All samples are derived from the same gel. However, some lanes of the gel were removed to make the data easier to interpret. p, precursor protein in the C/M fraction; m, mature MalE in the P fraction; asterisk, TorA-MalE-derived degradation products in the C/M fraction. The phenotypes of the respective strains on MMM (−: no growth; +: slow growth; ++: growth) and MCM (P: pale; LR: light red/pink; R: red) agar plates are shown in the boxes at the bottom of the figure.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3383694&req=5

pone-0039867-g001: Effect of mutations at the +2 position in the Tat consensus motif.Cells were fractionated into a periplasmic (P) and a combined cytosol/membrane fraction (C/M) by EDTA-lysozyme spheroplasting. The samples were subjected to SDS-PAGE and immunoblotting using anti-MalE antibodies. The positive control was E. coli GSJ101 containing plasmids pTorA-MalE and pHSG-TatABCE (lane 1). The other samples correspond to GSJ101 containing plasmid pHSG-TatABCE in addition to pTorA(S+2)-MalE (lane 2), pTorA(R+2)-MalE (lane 3), or pTorA(D+2)-MalE (lane 4). All samples are derived from the same gel. However, some lanes of the gel were removed to make the data easier to interpret. p, precursor protein in the C/M fraction; m, mature MalE in the P fraction; asterisk, TorA-MalE-derived degradation products in the C/M fraction. The phenotypes of the respective strains on MMM (−: no growth; +: slow growth; ++: growth) and MCM (P: pale; LR: light red/pink; R: red) agar plates are shown in the boxes at the bottom of the figure.

Mentions: To investigate the contribution of amino acid residues in the extended Tat motif other than the nearly invariable RR residues to signal peptide recognition by the Tat translocase, the phenylalanine at position +2 relative to the twin arginine residues was altered to serine, arginine, and aspartate, respectively. Like the wild-type control GSJ101 (pTorA-MalE, pHSG-TatABCE), also GSJ101 (pTorA(S+2)-MalE, pHSG-TatABCE) and GSJ101 (pTorA(R+2)-MalE, pHSG-TatABCE) were able to grow on MMM and formed red colonies on MCM agar plates, showing that the presence of either a serine or an arginine residue at the +2 position in the Tat motif does not preclude Tat-dependent export of the TorA-MalE reporter. In contrast, no growth on MMM and pale colonies on MCM agar plates were observed with GSJ101 (pTorA(D+2)-MalE, pHSG-TatABCE) (Figure 1). The export defect of TorA(D+2)-MalE was also directly visualized by determining the subcellular localization of MalE-derived polypeptides after EDTA-lysozyme spheroplasting in the corresponding cells. As shown in Figure 1, lane 1, upper part, several MalE-derived polypeptides are present in the combined fraction of cytosol and membranes (C/M) of GSJ101 (pTorA-MalE, pHSG-TatABCE), coexpressing the wild-type Tat translocase and the unaltered TorA-MalE (positive control). As described previously [17], these bands correspond to the unprocessed precursor protein and a variety of its cyosolic degradation products. In the periplasmic (P) fraction (Figure 1, lane 1, lower part), the mature-sized MalE is detected that has been translocated across the cytoplasmic membrane in a Tat-dependent manner [17]. In full accordance with the in situ phenotypes described above, in both GSJ101 coexpressing the Tat wild-type translocase and TorA(S+2)-MalE or TorA(R+2)-MalE respectively, mature MalE is present in the P fraction (Figure 1, lanes 2 and 3). In contrast, no mature MalE can be detected in the P fraction of GSJ101 coexpressing the wild-type Tat translocase and TorA(D+2)-MalE, showing that the negatively-charged aspartate is not tolerated at the +2 position in the extended twin-arginine motif and renders the TorA signal peptide defective for Tat-dependent protein translocation (Figure 1, lane 4).


Genetic evidence for a tight cooperation of TatB and TatC during productive recognition of twin-arginine (Tat) signal peptides in Escherichia coli.

Lausberg F, Fleckenstein S, Kreutzenbeck P, Fröbel J, Rose P, Müller M, Freudl R - PLoS ONE (2012)

Effect of mutations at the +2 position in the Tat consensus motif.Cells were fractionated into a periplasmic (P) and a combined cytosol/membrane fraction (C/M) by EDTA-lysozyme spheroplasting. The samples were subjected to SDS-PAGE and immunoblotting using anti-MalE antibodies. The positive control was E. coli GSJ101 containing plasmids pTorA-MalE and pHSG-TatABCE (lane 1). The other samples correspond to GSJ101 containing plasmid pHSG-TatABCE in addition to pTorA(S+2)-MalE (lane 2), pTorA(R+2)-MalE (lane 3), or pTorA(D+2)-MalE (lane 4). All samples are derived from the same gel. However, some lanes of the gel were removed to make the data easier to interpret. p, precursor protein in the C/M fraction; m, mature MalE in the P fraction; asterisk, TorA-MalE-derived degradation products in the C/M fraction. The phenotypes of the respective strains on MMM (−: no growth; +: slow growth; ++: growth) and MCM (P: pale; LR: light red/pink; R: red) agar plates are shown in the boxes at the bottom of the figure.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3383694&req=5

pone-0039867-g001: Effect of mutations at the +2 position in the Tat consensus motif.Cells were fractionated into a periplasmic (P) and a combined cytosol/membrane fraction (C/M) by EDTA-lysozyme spheroplasting. The samples were subjected to SDS-PAGE and immunoblotting using anti-MalE antibodies. The positive control was E. coli GSJ101 containing plasmids pTorA-MalE and pHSG-TatABCE (lane 1). The other samples correspond to GSJ101 containing plasmid pHSG-TatABCE in addition to pTorA(S+2)-MalE (lane 2), pTorA(R+2)-MalE (lane 3), or pTorA(D+2)-MalE (lane 4). All samples are derived from the same gel. However, some lanes of the gel were removed to make the data easier to interpret. p, precursor protein in the C/M fraction; m, mature MalE in the P fraction; asterisk, TorA-MalE-derived degradation products in the C/M fraction. The phenotypes of the respective strains on MMM (−: no growth; +: slow growth; ++: growth) and MCM (P: pale; LR: light red/pink; R: red) agar plates are shown in the boxes at the bottom of the figure.
Mentions: To investigate the contribution of amino acid residues in the extended Tat motif other than the nearly invariable RR residues to signal peptide recognition by the Tat translocase, the phenylalanine at position +2 relative to the twin arginine residues was altered to serine, arginine, and aspartate, respectively. Like the wild-type control GSJ101 (pTorA-MalE, pHSG-TatABCE), also GSJ101 (pTorA(S+2)-MalE, pHSG-TatABCE) and GSJ101 (pTorA(R+2)-MalE, pHSG-TatABCE) were able to grow on MMM and formed red colonies on MCM agar plates, showing that the presence of either a serine or an arginine residue at the +2 position in the Tat motif does not preclude Tat-dependent export of the TorA-MalE reporter. In contrast, no growth on MMM and pale colonies on MCM agar plates were observed with GSJ101 (pTorA(D+2)-MalE, pHSG-TatABCE) (Figure 1). The export defect of TorA(D+2)-MalE was also directly visualized by determining the subcellular localization of MalE-derived polypeptides after EDTA-lysozyme spheroplasting in the corresponding cells. As shown in Figure 1, lane 1, upper part, several MalE-derived polypeptides are present in the combined fraction of cytosol and membranes (C/M) of GSJ101 (pTorA-MalE, pHSG-TatABCE), coexpressing the wild-type Tat translocase and the unaltered TorA-MalE (positive control). As described previously [17], these bands correspond to the unprocessed precursor protein and a variety of its cyosolic degradation products. In the periplasmic (P) fraction (Figure 1, lane 1, lower part), the mature-sized MalE is detected that has been translocated across the cytoplasmic membrane in a Tat-dependent manner [17]. In full accordance with the in situ phenotypes described above, in both GSJ101 coexpressing the Tat wild-type translocase and TorA(S+2)-MalE or TorA(R+2)-MalE respectively, mature MalE is present in the P fraction (Figure 1, lanes 2 and 3). In contrast, no mature MalE can be detected in the P fraction of GSJ101 coexpressing the wild-type Tat translocase and TorA(D+2)-MalE, showing that the negatively-charged aspartate is not tolerated at the +2 position in the extended twin-arginine motif and renders the TorA signal peptide defective for Tat-dependent protein translocation (Figure 1, lane 4).

Bottom Line: Mutations were identified in the extreme amino-terminal regions of TatB and TatC that synergistically suppressed the export defect of TorA(D(+2))-MalE when present in pairwise or triple combinations.The observed synergistic suppression activities were even more pronounced in the restoration of membrane translocation of another export-defective precursor, TorA(KQ)-MalE, in which the conserved twin arginine residues had been replaced by lysine-glutamine.Collectively, these findings indicate that the extreme amino-terminal regions of TatB and TatC cooperate tightly during recognition and productive binding of Tat-dependent precursor proteins and, furthermore, that TatB and TatC are both involved in the formation of a specific signal peptide binding site that reaches out as far as the end of the TatB transmembrane segment.

View Article: PubMed Central - PubMed

Affiliation: Institut für Bio- und Geowissenschaften 1, Biotechnologie, Forschungszentrum Jülich GmbH, Jülich, Germany.

ABSTRACT
The twin arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane of bacteria. Tat signal peptides contain a consensus motif (S/T-R-R-X-F-L-K) that is thought to play a crucial role in substrate recognition by the Tat translocase. Replacement of the phenylalanine at the +2 consensus position in the signal peptide of a Tat-specific reporter protein (TorA-MalE) by aspartate blocked export of the corresponding TorA(D(+2))-MalE precursor, indicating that this mutation prevents a productive binding of the TorA(D(+2)) signal peptide to the Tat translocase. Mutations were identified in the extreme amino-terminal regions of TatB and TatC that synergistically suppressed the export defect of TorA(D(+2))-MalE when present in pairwise or triple combinations. The observed synergistic suppression activities were even more pronounced in the restoration of membrane translocation of another export-defective precursor, TorA(KQ)-MalE, in which the conserved twin arginine residues had been replaced by lysine-glutamine. Collectively, these findings indicate that the extreme amino-terminal regions of TatB and TatC cooperate tightly during recognition and productive binding of Tat-dependent precursor proteins and, furthermore, that TatB and TatC are both involved in the formation of a specific signal peptide binding site that reaches out as far as the end of the TatB transmembrane segment.

Show MeSH
Related in: MedlinePlus