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Initial assembly steps of a translocase for folded proteins.

Blümmel AS, Haag LA, Eimer E, Müller M, Fröbel J - Nat Commun (2015)

Bottom Line: Many Tat systems are based on the membrane proteins TatA, TatB and TatC, of which TatB and TatC are known to cooperate in binding RR-signal peptides and to form higher-order oligomeric structures.The identification of distinct homonymous and heteronymous contacts between TatB and TatC suggest that TatB monomers coalesce into dome-like TatB structures that are surrounded by outer rings of TatC monomers.We also show that these TatBC complexes are approached by TatA protomers through their N-termini, which thereby establish contacts with TatB and membrane-inserted RR-precursors.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Biochemistry and Molecular Biology, ZBMZ, University of Freiburg, 79104 Freiburg, Germany [2] Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, 79104 Freiburg, Germany [3] Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany.

ABSTRACT
The so-called Tat (twin-arginine translocation) system transports completely folded proteins across cellular membranes of archaea, prokaryotes and plant chloroplasts. Tat-directed proteins are distinguished by a conserved twin-arginine (RR-) motif in their signal sequences. Many Tat systems are based on the membrane proteins TatA, TatB and TatC, of which TatB and TatC are known to cooperate in binding RR-signal peptides and to form higher-order oligomeric structures. We have now elucidated the fine architecture of TatBC oligomers assembled to form closed intramembrane substrate-binding cavities. The identification of distinct homonymous and heteronymous contacts between TatB and TatC suggest that TatB monomers coalesce into dome-like TatB structures that are surrounded by outer rings of TatC monomers. We also show that these TatBC complexes are approached by TatA protomers through their N-termini, which thereby establish contacts with TatB and membrane-inserted RR-precursors.

No MeSH data available.


Related in: MedlinePlus

A TatBC-walled insertion site for RR-precursors.(a) N-terminal sequence shared by the model RR-precursors TorA-MalE and TorA-mCherry with the residues highlighted that were replaced by Bpa. The six amino acids flanking the cleavage site (slash) of the TorA signal peptide are underlined. (b) The indicated Bpa variants of TorA-MalE (TMal) were synthesized in vitro in the presence of membrane vesicles containing the TatABC proteins and crosslinking was initiated by ultraviolet light irradiation (for details see legend to Fig. 1b). One aliquot of each irradiated reaction (+) was directly analysed by SDS–PAGE and phosphorimaging (lanes 2, 7, 12, 17, 23, 28), while others were first immuno-precipitated (IP) using antibodies against TatA, TatB, TatC (lanes A,B,C). Crosslinking products recognized by anti-TatB and anti-TatC antibodies are indicated by the green and blue stars, respectively. No TatA-immuno-reactive adducts were obtained. (c) The P34Bpa variant of TorA-mCherry (TmC) was synthesized in vitro in the presence of membrane vesicles (INV) containing the indicated Tat proteins. Crosslinking and labelling of the adducts are as in b.
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f3: A TatBC-walled insertion site for RR-precursors.(a) N-terminal sequence shared by the model RR-precursors TorA-MalE and TorA-mCherry with the residues highlighted that were replaced by Bpa. The six amino acids flanking the cleavage site (slash) of the TorA signal peptide are underlined. (b) The indicated Bpa variants of TorA-MalE (TMal) were synthesized in vitro in the presence of membrane vesicles containing the TatABC proteins and crosslinking was initiated by ultraviolet light irradiation (for details see legend to Fig. 1b). One aliquot of each irradiated reaction (+) was directly analysed by SDS–PAGE and phosphorimaging (lanes 2, 7, 12, 17, 23, 28), while others were first immuno-precipitated (IP) using antibodies against TatA, TatB, TatC (lanes A,B,C). Crosslinking products recognized by anti-TatB and anti-TatC antibodies are indicated by the green and blue stars, respectively. No TatA-immuno-reactive adducts were obtained. (c) The P34Bpa variant of TorA-mCherry (TmC) was synthesized in vitro in the presence of membrane vesicles (INV) containing the indicated Tat proteins. Crosslinking and labelling of the adducts are as in b.

Mentions: As noted above, the TM5 of TatC seems to be a common docking site for RR-precursors and TatB raising the question of a direct contact between TatB and RR-precursors in this trans-sided area of TatC. If so, TatB might shield the cleavage site of a deeply inserted RR-precursor and thereby prevent premature exposure to signal peptidase as recently suggested18. To experimentally verify such a juxtaposition of RR-precursor and TatB, we incorporated Bpa into the TorA signal sequence at several positions reaching from the RR-consensus motif until beyond the cleavage site (Fig. 3a). The Bpa variants of the model Tat substrate TorA-MalE19 were synthesized in vitro in the presence of INV containing all three Tat subunits and crosslinking was initiated by irradiation with ultraviolet light (Fig. 3b). When inserted at position F14, Bpa crosslinked TorA-MalE (TMal) to TatC as verified by immuno-precipitation of the crosslinking products (Fig. 3b; lanes 2 and 5, blue stars). This confirms the previously established recognition of the RR-containing consensus motif by TatC202526. In addition to the ∼60 kDa TatC-TMal adduct, an ∼90 kDa ultraviolet light-dependent crosslinking product was recognized by the anti-TatC antibodies (Fig. 3b; lanes 2 and 5, upper blue star) suggesting that two closely spaced precursors27 contact one TatC molecule. In contrast, all the sites selected further downstream in the TorA signal sequence (V23, L27, P34) and beyond the signal sequence cleavage site (V47 and F49) crosslinked to TatB (Fig. 3b; green stars). For the V47Bpa variant of TMal, an additional larger TatB-cross-reactive adduct was obtained (lane 25) that could reflect binding of two precursors to the same TatB monomer either individually or as a linked dimer. If the Bpa variants of the TorA signal sequence were incubated with INV that selectively lacked TatB, the originally TatB-reactive sites were now found in proximity to TatC. This is demonstrated in Fig. 3c for the P34Bpa variant of TorA-mCherry (TmC), which yielded a prominent adduct of the size of a TatB–TmC complex whenever TatB was present (lanes 2 and 6, green stars). In its absence, however, the smaller adduct to TatC was obtained (lanes 4 and 8, blue stars). These results are consistent with a TatBC-walled insertion cavity, in which the TorA signal sequence would be closer to TatB so that interactions with the more external TatC become detectable only in the absence of TatB.


Initial assembly steps of a translocase for folded proteins.

Blümmel AS, Haag LA, Eimer E, Müller M, Fröbel J - Nat Commun (2015)

A TatBC-walled insertion site for RR-precursors.(a) N-terminal sequence shared by the model RR-precursors TorA-MalE and TorA-mCherry with the residues highlighted that were replaced by Bpa. The six amino acids flanking the cleavage site (slash) of the TorA signal peptide are underlined. (b) The indicated Bpa variants of TorA-MalE (TMal) were synthesized in vitro in the presence of membrane vesicles containing the TatABC proteins and crosslinking was initiated by ultraviolet light irradiation (for details see legend to Fig. 1b). One aliquot of each irradiated reaction (+) was directly analysed by SDS–PAGE and phosphorimaging (lanes 2, 7, 12, 17, 23, 28), while others were first immuno-precipitated (IP) using antibodies against TatA, TatB, TatC (lanes A,B,C). Crosslinking products recognized by anti-TatB and anti-TatC antibodies are indicated by the green and blue stars, respectively. No TatA-immuno-reactive adducts were obtained. (c) The P34Bpa variant of TorA-mCherry (TmC) was synthesized in vitro in the presence of membrane vesicles (INV) containing the indicated Tat proteins. Crosslinking and labelling of the adducts are as in b.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490388&req=5

f3: A TatBC-walled insertion site for RR-precursors.(a) N-terminal sequence shared by the model RR-precursors TorA-MalE and TorA-mCherry with the residues highlighted that were replaced by Bpa. The six amino acids flanking the cleavage site (slash) of the TorA signal peptide are underlined. (b) The indicated Bpa variants of TorA-MalE (TMal) were synthesized in vitro in the presence of membrane vesicles containing the TatABC proteins and crosslinking was initiated by ultraviolet light irradiation (for details see legend to Fig. 1b). One aliquot of each irradiated reaction (+) was directly analysed by SDS–PAGE and phosphorimaging (lanes 2, 7, 12, 17, 23, 28), while others were first immuno-precipitated (IP) using antibodies against TatA, TatB, TatC (lanes A,B,C). Crosslinking products recognized by anti-TatB and anti-TatC antibodies are indicated by the green and blue stars, respectively. No TatA-immuno-reactive adducts were obtained. (c) The P34Bpa variant of TorA-mCherry (TmC) was synthesized in vitro in the presence of membrane vesicles (INV) containing the indicated Tat proteins. Crosslinking and labelling of the adducts are as in b.
Mentions: As noted above, the TM5 of TatC seems to be a common docking site for RR-precursors and TatB raising the question of a direct contact between TatB and RR-precursors in this trans-sided area of TatC. If so, TatB might shield the cleavage site of a deeply inserted RR-precursor and thereby prevent premature exposure to signal peptidase as recently suggested18. To experimentally verify such a juxtaposition of RR-precursor and TatB, we incorporated Bpa into the TorA signal sequence at several positions reaching from the RR-consensus motif until beyond the cleavage site (Fig. 3a). The Bpa variants of the model Tat substrate TorA-MalE19 were synthesized in vitro in the presence of INV containing all three Tat subunits and crosslinking was initiated by irradiation with ultraviolet light (Fig. 3b). When inserted at position F14, Bpa crosslinked TorA-MalE (TMal) to TatC as verified by immuno-precipitation of the crosslinking products (Fig. 3b; lanes 2 and 5, blue stars). This confirms the previously established recognition of the RR-containing consensus motif by TatC202526. In addition to the ∼60 kDa TatC-TMal adduct, an ∼90 kDa ultraviolet light-dependent crosslinking product was recognized by the anti-TatC antibodies (Fig. 3b; lanes 2 and 5, upper blue star) suggesting that two closely spaced precursors27 contact one TatC molecule. In contrast, all the sites selected further downstream in the TorA signal sequence (V23, L27, P34) and beyond the signal sequence cleavage site (V47 and F49) crosslinked to TatB (Fig. 3b; green stars). For the V47Bpa variant of TMal, an additional larger TatB-cross-reactive adduct was obtained (lane 25) that could reflect binding of two precursors to the same TatB monomer either individually or as a linked dimer. If the Bpa variants of the TorA signal sequence were incubated with INV that selectively lacked TatB, the originally TatB-reactive sites were now found in proximity to TatC. This is demonstrated in Fig. 3c for the P34Bpa variant of TorA-mCherry (TmC), which yielded a prominent adduct of the size of a TatB–TmC complex whenever TatB was present (lanes 2 and 6, green stars). In its absence, however, the smaller adduct to TatC was obtained (lanes 4 and 8, blue stars). These results are consistent with a TatBC-walled insertion cavity, in which the TorA signal sequence would be closer to TatB so that interactions with the more external TatC become detectable only in the absence of TatB.

Bottom Line: Many Tat systems are based on the membrane proteins TatA, TatB and TatC, of which TatB and TatC are known to cooperate in binding RR-signal peptides and to form higher-order oligomeric structures.The identification of distinct homonymous and heteronymous contacts between TatB and TatC suggest that TatB monomers coalesce into dome-like TatB structures that are surrounded by outer rings of TatC monomers.We also show that these TatBC complexes are approached by TatA protomers through their N-termini, which thereby establish contacts with TatB and membrane-inserted RR-precursors.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Biochemistry and Molecular Biology, ZBMZ, University of Freiburg, 79104 Freiburg, Germany [2] Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, 79104 Freiburg, Germany [3] Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany.

ABSTRACT
The so-called Tat (twin-arginine translocation) system transports completely folded proteins across cellular membranes of archaea, prokaryotes and plant chloroplasts. Tat-directed proteins are distinguished by a conserved twin-arginine (RR-) motif in their signal sequences. Many Tat systems are based on the membrane proteins TatA, TatB and TatC, of which TatB and TatC are known to cooperate in binding RR-signal peptides and to form higher-order oligomeric structures. We have now elucidated the fine architecture of TatBC oligomers assembled to form closed intramembrane substrate-binding cavities. The identification of distinct homonymous and heteronymous contacts between TatB and TatC suggest that TatB monomers coalesce into dome-like TatB structures that are surrounded by outer rings of TatC monomers. We also show that these TatBC complexes are approached by TatA protomers through their N-termini, which thereby establish contacts with TatB and membrane-inserted RR-precursors.

No MeSH data available.


Related in: MedlinePlus