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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

Contacts between TatC and a membrane-inserted RR-precursor.(a) Model of E. coli TatC based on the structure of A. aeolicus TatC7 using PDB code 4B4A. Indicated in yellow are all amino acids replaced by Bpa in this study, and in orange those of a previous analysis19. (b) The model RR-precursor TorA-mCherry was synthesized and radioactively labelled by in vitro transcription/translation in the absence or presence of inverted E. coli inner membrane vesicles (INV). In addition to TatA and TatB, INV contained either wild-type TatC (TatABC) or the indicated Bpa variants of TatC (TatABCBpa). In samples labelled (+), crosslinking was initiated by irradiation with ultraviolet light. Radiolabelled translation products were separated by SDS–PAGE and visualized by phosphorimaging. Indicated are the positions of molecular size standard proteins (left-hand side), the TorA-mCherry (TmC) precursor (p), and the crosslinked TatC–TmC complex (blue star). The black star marks a ultraviolet light-dependent crosslink of unknown nature between TorA-mCherry and several TatC variants. (c) comparing crosslinking of the L206Bpa variant of TatC to wild-type TorA-mCherry (RR) and to a mutant with an inactive signal peptide (KK). (d) Highlighted in red are all residues that in b showed distinct contacts to TorA-mCherry when replaced by Bpa, and in blue the precursor contact sites identified in our previous study19. The helices of TatC are numbered.
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f1: Contacts between TatC and a membrane-inserted RR-precursor.(a) Model of E. coli TatC based on the structure of A. aeolicus TatC7 using PDB code 4B4A. Indicated in yellow are all amino acids replaced by Bpa in this study, and in orange those of a previous analysis19. (b) The model RR-precursor TorA-mCherry was synthesized and radioactively labelled by in vitro transcription/translation in the absence or presence of inverted E. coli inner membrane vesicles (INV). In addition to TatA and TatB, INV contained either wild-type TatC (TatABC) or the indicated Bpa variants of TatC (TatABCBpa). In samples labelled (+), crosslinking was initiated by irradiation with ultraviolet light. Radiolabelled translation products were separated by SDS–PAGE and visualized by phosphorimaging. Indicated are the positions of molecular size standard proteins (left-hand side), the TorA-mCherry (TmC) precursor (p), and the crosslinked TatC–TmC complex (blue star). The black star marks a ultraviolet light-dependent crosslink of unknown nature between TorA-mCherry and several TatC variants. (c) comparing crosslinking of the L206Bpa variant of TatC to wild-type TorA-mCherry (RR) and to a mutant with an inactive signal peptide (KK). (d) Highlighted in red are all residues that in b showed distinct contacts to TorA-mCherry when replaced by Bpa, and in blue the precursor contact sites identified in our previous study19. The helices of TatC are numbered.

Mentions: We recently demonstrated that in the absence of TatB, TatC is sufficient to insert RR-precursor proteins into the plasma membrane of E. coli even to a point where they become prematurely processed by signal peptidase18. We now sought to follow the track of such an inserted RR-precursor on the TatC molecule. To this end, we incorporated the photo-activatable crosslinker p-benzoyl-phenylalanine (Bpa) into 28 novel positions of E. coli TatC (Fig. 1a; shown in yellow) that had not been analysed previously19 (Fig. 1a; orange residues). Inside-out inner-membrane vesicles (INV) were prepared from E. coli strains expressing the individual Bpa-containing variants of TatC and were incubated with the in vitro synthesized and radioactively labelled Tat model substrate TorA-mCherry18. Results obtained with 10 of the TatC variants are shown in Fig. 1b. Ultraviolet light-dependent, radiolabelled crosslinking products of the size of 1:1 complexes between TorA-mCherry and TatC (55 kDa, blue star) appeared when Bpa had been incorporated at the positions 181, 202, 205, 206 and 208 of TatC. The specificity of these TatC–TorA-mCherry contacts is indicated by the failure to obtain them when using a transport-incompetent KK-mutant of TorA-mCherry (Fig. 1c). Several residues gave rise to ultraviolet light-dependent crosslinks that were ∼10 kDa larger (Fig. 1b; black star). They could not clearly be distinguished from an unspecific band and therefore remain of unknown origin at this point.


Initial assembly steps of a translocase for folded proteins.

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

Contacts between TatC and a membrane-inserted RR-precursor.(a) Model of E. coli TatC based on the structure of A. aeolicus TatC7 using PDB code 4B4A. Indicated in yellow are all amino acids replaced by Bpa in this study, and in orange those of a previous analysis19. (b) The model RR-precursor TorA-mCherry was synthesized and radioactively labelled by in vitro transcription/translation in the absence or presence of inverted E. coli inner membrane vesicles (INV). In addition to TatA and TatB, INV contained either wild-type TatC (TatABC) or the indicated Bpa variants of TatC (TatABCBpa). In samples labelled (+), crosslinking was initiated by irradiation with ultraviolet light. Radiolabelled translation products were separated by SDS–PAGE and visualized by phosphorimaging. Indicated are the positions of molecular size standard proteins (left-hand side), the TorA-mCherry (TmC) precursor (p), and the crosslinked TatC–TmC complex (blue star). The black star marks a ultraviolet light-dependent crosslink of unknown nature between TorA-mCherry and several TatC variants. (c) comparing crosslinking of the L206Bpa variant of TatC to wild-type TorA-mCherry (RR) and to a mutant with an inactive signal peptide (KK). (d) Highlighted in red are all residues that in b showed distinct contacts to TorA-mCherry when replaced by Bpa, and in blue the precursor contact sites identified in our previous study19. The helices of TatC are numbered.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Contacts between TatC and a membrane-inserted RR-precursor.(a) Model of E. coli TatC based on the structure of A. aeolicus TatC7 using PDB code 4B4A. Indicated in yellow are all amino acids replaced by Bpa in this study, and in orange those of a previous analysis19. (b) The model RR-precursor TorA-mCherry was synthesized and radioactively labelled by in vitro transcription/translation in the absence or presence of inverted E. coli inner membrane vesicles (INV). In addition to TatA and TatB, INV contained either wild-type TatC (TatABC) or the indicated Bpa variants of TatC (TatABCBpa). In samples labelled (+), crosslinking was initiated by irradiation with ultraviolet light. Radiolabelled translation products were separated by SDS–PAGE and visualized by phosphorimaging. Indicated are the positions of molecular size standard proteins (left-hand side), the TorA-mCherry (TmC) precursor (p), and the crosslinked TatC–TmC complex (blue star). The black star marks a ultraviolet light-dependent crosslink of unknown nature between TorA-mCherry and several TatC variants. (c) comparing crosslinking of the L206Bpa variant of TatC to wild-type TorA-mCherry (RR) and to a mutant with an inactive signal peptide (KK). (d) Highlighted in red are all residues that in b showed distinct contacts to TorA-mCherry when replaced by Bpa, and in blue the precursor contact sites identified in our previous study19. The helices of TatC are numbered.
Mentions: We recently demonstrated that in the absence of TatB, TatC is sufficient to insert RR-precursor proteins into the plasma membrane of E. coli even to a point where they become prematurely processed by signal peptidase18. We now sought to follow the track of such an inserted RR-precursor on the TatC molecule. To this end, we incorporated the photo-activatable crosslinker p-benzoyl-phenylalanine (Bpa) into 28 novel positions of E. coli TatC (Fig. 1a; shown in yellow) that had not been analysed previously19 (Fig. 1a; orange residues). Inside-out inner-membrane vesicles (INV) were prepared from E. coli strains expressing the individual Bpa-containing variants of TatC and were incubated with the in vitro synthesized and radioactively labelled Tat model substrate TorA-mCherry18. Results obtained with 10 of the TatC variants are shown in Fig. 1b. Ultraviolet light-dependent, radiolabelled crosslinking products of the size of 1:1 complexes between TorA-mCherry and TatC (55 kDa, blue star) appeared when Bpa had been incorporated at the positions 181, 202, 205, 206 and 208 of TatC. The specificity of these TatC–TorA-mCherry contacts is indicated by the failure to obtain them when using a transport-incompetent KK-mutant of TorA-mCherry (Fig. 1c). Several residues gave rise to ultraviolet light-dependent crosslinks that were ∼10 kDa larger (Fig. 1b; black star). They could not clearly be distinguished from an unspecific band and therefore remain of unknown origin at this point.

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