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translocation of another export-defective precursor, TorA(KQ)-MalE, in which the conserved twin arginine residues had been replaced by lysine-glutamine.Collectively, these findings indicate that the extreme amino-terminal regions of TatB and TatC cooperate tightly during recognition and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translocation of another export-defective precursor, TorA(KQ)-MalE, in which the conserved twin arginine residues had been replaced by lysine-glutamine.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

Subcellular localization of TorA(D+2)-MalE-derived polypeptides.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 (lanes 1). All other samples correspond to GSJ101 containing plasmid pTorA(D+2)-MalE in addition to a pHSG-TatABCE plasmid that encodes one of the mutant translocases derived from RRD2 (A), RRD3 (B), RRD1 (C), or the synthetic mutant translocases RRD5/6 (D), as indicated above the lanes. p, TorA-MalE/TorA(D+2)-MalE precursor in the C/M fraction; m, mature MalE in the P fraction; asterisk, TorA-MalE/TorA(D+2)-MalE degradation products in the C/M fraction. All samples shown in the respective panels are derived from the same gel. However, in some cases lanes of the gels were removed to make the data easier to interpret. 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. The nature of the signal peptide (SP) of the respective TorA-MalE precursors (wild-type (Wt) or containing the D+2 mutation (D+2)) and the TatB and/or TatC mutations present in the respective translocases are indicated in the boxes at the top of the panels. E. Relative export efficiencies. The amount of MalE in the P fraction of strains expressing TorA-MalE or TorA(D+2)-MalE in combination with the Tat translocases indicated below the bars was determined in at least three independent experiments via quantification of the chemiluminescence signals. The signals were recorded by a CCD camera and subsequently analyzed by the program AIDA 4.15 (Raytest). %, average value of relative export efficiency. The relative export efficiency of the respective positive control strain GSJ101 (pTorA-MalE, pHSG-TatABCE) in each experiment (lane 1 in panels A–D) was set to 100%.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3383694&req=5

pone-0039867-g003: Subcellular localization of TorA(D+2)-MalE-derived polypeptides.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 (lanes 1). All other samples correspond to GSJ101 containing plasmid pTorA(D+2)-MalE in addition to a pHSG-TatABCE plasmid that encodes one of the mutant translocases derived from RRD2 (A), RRD3 (B), RRD1 (C), or the synthetic mutant translocases RRD5/6 (D), as indicated above the lanes. p, TorA-MalE/TorA(D+2)-MalE precursor in the C/M fraction; m, mature MalE in the P fraction; asterisk, TorA-MalE/TorA(D+2)-MalE degradation products in the C/M fraction. All samples shown in the respective panels are derived from the same gel. However, in some cases lanes of the gels were removed to make the data easier to interpret. 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. The nature of the signal peptide (SP) of the respective TorA-MalE precursors (wild-type (Wt) or containing the D+2 mutation (D+2)) and the TatB and/or TatC mutations present in the respective translocases are indicated in the boxes at the top of the panels. E. Relative export efficiencies. The amount of MalE in the P fraction of strains expressing TorA-MalE or TorA(D+2)-MalE in combination with the Tat translocases indicated below the bars was determined in at least three independent experiments via quantification of the chemiluminescence signals. The signals were recorded by a CCD camera and subsequently analyzed by the program AIDA 4.15 (Raytest). %, average value of relative export efficiency. The relative export efficiency of the respective positive control strain GSJ101 (pTorA-MalE, pHSG-TatABCE) in each experiment (lane 1 in panels A–D) was set to 100%.

Mentions: TorA(D+2)-MalE export in the strains expressing the various Tat mutant translocases was analyzed indirectly by MMM and MCM plate assays and directly by determining the amount of MalE in the periplasm (Figure 3). The relative export efficiency (reflected by the amount of mature-sized MalE present in the periplasm) of the positive control strain, to which all further relative export efficiencies described in this work will be related, was set to 100%. All numbers indicated in the following represent average relative export efficiencies obtained from at least three independent experiments. From these combined analyses, it became evident that in all three mutant isolates a synergistically acting combination of two mutations (i.e. the L9F mutation in TatC together with a newly selected mutation in either TatC or TatB) is responsible for the suppression of the TorA(D+2)-MalE export defect.


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)

Subcellular localization of TorA(D+2)-MalE-derived polypeptides.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 (lanes 1). All other samples correspond to GSJ101 containing plasmid pTorA(D+2)-MalE in addition to a pHSG-TatABCE plasmid that encodes one of the mutant translocases derived from RRD2 (A), RRD3 (B), RRD1 (C), or the synthetic mutant translocases RRD5/6 (D), as indicated above the lanes. p, TorA-MalE/TorA(D+2)-MalE precursor in the C/M fraction; m, mature MalE in the P fraction; asterisk, TorA-MalE/TorA(D+2)-MalE degradation products in the C/M fraction. All samples shown in the respective panels are derived from the same gel. However, in some cases lanes of the gels were removed to make the data easier to interpret. 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. The nature of the signal peptide (SP) of the respective TorA-MalE precursors (wild-type (Wt) or containing the D+2 mutation (D+2)) and the TatB and/or TatC mutations present in the respective translocases are indicated in the boxes at the top of the panels. E. Relative export efficiencies. The amount of MalE in the P fraction of strains expressing TorA-MalE or TorA(D+2)-MalE in combination with the Tat translocases indicated below the bars was determined in at least three independent experiments via quantification of the chemiluminescence signals. The signals were recorded by a CCD camera and subsequently analyzed by the program AIDA 4.15 (Raytest). %, average value of relative export efficiency. The relative export efficiency of the respective positive control strain GSJ101 (pTorA-MalE, pHSG-TatABCE) in each experiment (lane 1 in panels A–D) was set to 100%.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0039867-g003: Subcellular localization of TorA(D+2)-MalE-derived polypeptides.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 (lanes 1). All other samples correspond to GSJ101 containing plasmid pTorA(D+2)-MalE in addition to a pHSG-TatABCE plasmid that encodes one of the mutant translocases derived from RRD2 (A), RRD3 (B), RRD1 (C), or the synthetic mutant translocases RRD5/6 (D), as indicated above the lanes. p, TorA-MalE/TorA(D+2)-MalE precursor in the C/M fraction; m, mature MalE in the P fraction; asterisk, TorA-MalE/TorA(D+2)-MalE degradation products in the C/M fraction. All samples shown in the respective panels are derived from the same gel. However, in some cases lanes of the gels were removed to make the data easier to interpret. 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. The nature of the signal peptide (SP) of the respective TorA-MalE precursors (wild-type (Wt) or containing the D+2 mutation (D+2)) and the TatB and/or TatC mutations present in the respective translocases are indicated in the boxes at the top of the panels. E. Relative export efficiencies. The amount of MalE in the P fraction of strains expressing TorA-MalE or TorA(D+2)-MalE in combination with the Tat translocases indicated below the bars was determined in at least three independent experiments via quantification of the chemiluminescence signals. The signals were recorded by a CCD camera and subsequently analyzed by the program AIDA 4.15 (Raytest). %, average value of relative export efficiency. The relative export efficiency of the respective positive control strain GSJ101 (pTorA-MalE, pHSG-TatABCE) in each experiment (lane 1 in panels A–D) was set to 100%.
Mentions: TorA(D+2)-MalE export in the strains expressing the various Tat mutant translocases was analyzed indirectly by MMM and MCM plate assays and directly by determining the amount of MalE in the periplasm (Figure 3). The relative export efficiency (reflected by the amount of mature-sized MalE present in the periplasm) of the positive control strain, to which all further relative export efficiencies described in this work will be related, was set to 100%. All numbers indicated in the following represent average relative export efficiencies obtained from at least three independent experiments. From these combined analyses, it became evident that in all three mutant isolates a synergistically acting combination of two mutations (i.e. the L9F mutation in TatC together with a newly selected mutation in either TatC or TatB) is responsible for the suppression of the TorA(D+2)-MalE export defect.

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