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translocation of another export-defective precursor, TorA(KQ)-MalE, in which the conserved twin arginine residues had been replaced by lysine-glutamine.Collectively, these findings indicate that the extreme amino-terminal regions of TatB and TatC cooperate tightly during recognition and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translocation of another export-defective precursor, TorA(KQ)-MalE, in which the conserved twin arginine residues had been replaced by lysine-glutamine.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

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Membrane topology of E. coli TatB and TatC and positions of mutations.Arrows indicate the positions of mutations that are involved in the suppression of the TorA(D+2)-MalE and TorA(KQ)-MalE export defects.
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pone-0039867-g002: Membrane topology of E. coli TatB and TatC and positions of mutations.Arrows indicate the positions of mutations that are involved in the suppression of the TorA(D+2)-MalE and TorA(KQ)-MalE export defects.

Mentions: DNA sequencing of the corresponding plasmids (pHSG-TatABCE-RRD1 to 3) showed that, in addition to the L9F mutation in TatC, multiple mutations are present in the respective mutant Tat translocases (Table 2). Interestingly, similar to the previously described KQS mutant Tat translocases that allowed export of the TorA(KQ)-MalE precursor [13], in each of the mutant translocases (designated RRD1 to 3), some of the newly selected mutations present in the multiple mutants map to the extreme amino-terminal ends of TatB and TatC (Table 2, Figure 2). Subsequently, these mutations were analyzed with respect to their contribution to the suppressing activity by introducing them alone or in pairwise combinations into otherwise wild-type tat genes (Table 2, and see below). Furthermore, the amounts of TatA, TatB and TatC proteins, present in the membrane fractions of the strains expressing these specifically constructed Tat translocases, were analyzed by Western blotting (Figure S2A). In most cases, the amounts of the Tat components were found to be similar or somewhat lower compared to the wild-type control. A noticeable difference, however, was found for TatB containing the mutation L9P. Here, the mutant TatB protein was only detected in the membrane after over-exposure of the Western blot (Figure S2B), suggesting that the respective TatB protein is relatively unstable and proteolytically degraded. Nevertheless, the mutant TatB protein is unequivocally required for the suppressing activity of the corresponding mutant translocases, since a Tat translocase lacking TatB did not show export of the TorA(D+2)-MalE mutant precursor (data not shown). Importantly, however, these findings exclude the possibility that the observed gain-of-function phenotypes, conferred by the mutant Tat translocases, are simply due to increased amounts of one or more Tat proteins and are in fact caused by the corresponding mutations.


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)

Membrane topology of E. coli TatB and TatC and positions of mutations.Arrows indicate the positions of mutations that are involved in the suppression of the TorA(D+2)-MalE and TorA(KQ)-MalE export defects.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0039867-g002: Membrane topology of E. coli TatB and TatC and positions of mutations.Arrows indicate the positions of mutations that are involved in the suppression of the TorA(D+2)-MalE and TorA(KQ)-MalE export defects.
Mentions: DNA sequencing of the corresponding plasmids (pHSG-TatABCE-RRD1 to 3) showed that, in addition to the L9F mutation in TatC, multiple mutations are present in the respective mutant Tat translocases (Table 2). Interestingly, similar to the previously described KQS mutant Tat translocases that allowed export of the TorA(KQ)-MalE precursor [13], in each of the mutant translocases (designated RRD1 to 3), some of the newly selected mutations present in the multiple mutants map to the extreme amino-terminal ends of TatB and TatC (Table 2, Figure 2). Subsequently, these mutations were analyzed with respect to their contribution to the suppressing activity by introducing them alone or in pairwise combinations into otherwise wild-type tat genes (Table 2, and see below). Furthermore, the amounts of TatA, TatB and TatC proteins, present in the membrane fractions of the strains expressing these specifically constructed Tat translocases, were analyzed by Western blotting (Figure S2A). In most cases, the amounts of the Tat components were found to be similar or somewhat lower compared to the wild-type control. A noticeable difference, however, was found for TatB containing the mutation L9P. Here, the mutant TatB protein was only detected in the membrane after over-exposure of the Western blot (Figure S2B), suggesting that the respective TatB protein is relatively unstable and proteolytically degraded. Nevertheless, the mutant TatB protein is unequivocally required for the suppressing activity of the corresponding mutant translocases, since a Tat translocase lacking TatB did not show export of the TorA(D+2)-MalE mutant precursor (data not shown). Importantly, however, these findings exclude the possibility that the observed gain-of-function phenotypes, conferred by the mutant Tat translocases, are simply due to increased amounts of one or more Tat proteins and are in fact caused by the corresponding mutations.

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