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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

Membrane-inserted RR-precursors touch at the N-termini of oligomeric TatB, subsequently also of TatA.(a) In vitro synthesis of the model RR-precursor TorA-mCherry in the presence of membrane vesicles (INV) containing wild-type TatABC, TatBC plus the G3Bpa variant of TatA and TatAC plus the F6Bpa variant of TatB. Crosslinking between these N-terminal Bpa variants and TorA-mCherry was initiated by ultraviolet light irradiation (+). Adducts of TorA-mCherry (TmC) to TatA (red stars) and TatB (green stars) could be purified via the His-tag of TorA-mCherry (His). The positions of the precursor (p) and the signal sequence-less form (m) of TorA-mCherry are indicated. (b) As in a, comparing crosslinking of the N-termini of TatA and TatB to wild-type TorA-mCherry (RR) and to its transport-incompetent mutant (KK). (c) As in a, using the three indicated N-terminal Bpa variants of TatA and TatB and the natural E. coli Tat substrate pSufI. In addition, the effects of dissipating the H+-motive force by the uncoupler CCCP on the crosslinking behaviour of pSufI to the N-termini of TatA and TatB are shown (red and green stars). The effectivity of CCCP is demonstrated by the failure of TatABC-INV (and others) to process the precursor of SufI (pSufI) to the signal sequence-less mature form (mSufI; lane 3).
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f4: Membrane-inserted RR-precursors touch at the N-termini of oligomeric TatB, subsequently also of TatA.(a) In vitro synthesis of the model RR-precursor TorA-mCherry in the presence of membrane vesicles (INV) containing wild-type TatABC, TatBC plus the G3Bpa variant of TatA and TatAC plus the F6Bpa variant of TatB. Crosslinking between these N-terminal Bpa variants and TorA-mCherry was initiated by ultraviolet light irradiation (+). Adducts of TorA-mCherry (TmC) to TatA (red stars) and TatB (green stars) could be purified via the His-tag of TorA-mCherry (His). The positions of the precursor (p) and the signal sequence-less form (m) of TorA-mCherry are indicated. (b) As in a, comparing crosslinking of the N-termini of TatA and TatB to wild-type TorA-mCherry (RR) and to its transport-incompetent mutant (KK). (c) As in a, using the three indicated N-terminal Bpa variants of TatA and TatB and the natural E. coli Tat substrate pSufI. In addition, the effects of dissipating the H+-motive force by the uncoupler CCCP on the crosslinking behaviour of pSufI to the N-termini of TatA and TatB are shown (red and green stars). The effectivity of CCCP is demonstrated by the failure of TatABC-INV (and others) to process the precursor of SufI (pSufI) to the signal sequence-less mature form (mSufI; lane 3).

Mentions: Next, we incubated INV that contained N-terminal Bpa variants of TatA or TatB with in vitro synthesized and radioactively labelled Tat substrates. In full agreement with a deep insertion, ultraviolet light-dependent adducts of TorA-mCherry to both N-termini of TatA and TatB (Fig. 4a; red and green stars) were observed. Notably, multiple adducts were obtained in either case. Because all of them were radioactively labelled and could be purified through the His-tag attached to TorA-mCherry (lanes 6 and 9), they all must contain the RR-precursor. Those multiple contacts are functionally relevant, because they were all abrogated when a transport-incompetent KK-mutant of TorA-mCherry was used (Fig. 4b). Very similar results were obtained for the natural E. coli Tat substrate pSufI (pFtsP) and three N-terminal Bpa variants of TatA and TatB each (Fig. 4c). These results clearly indicate that after insertion into the Tat translocase, RR-precursor molecules can associate simultaneously with the N-termini of multiple TatB and TatA molecules.


Initial assembly steps of a translocase for folded proteins.

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

Membrane-inserted RR-precursors touch at the N-termini of oligomeric TatB, subsequently also of TatA.(a) In vitro synthesis of the model RR-precursor TorA-mCherry in the presence of membrane vesicles (INV) containing wild-type TatABC, TatBC plus the G3Bpa variant of TatA and TatAC plus the F6Bpa variant of TatB. Crosslinking between these N-terminal Bpa variants and TorA-mCherry was initiated by ultraviolet light irradiation (+). Adducts of TorA-mCherry (TmC) to TatA (red stars) and TatB (green stars) could be purified via the His-tag of TorA-mCherry (His). The positions of the precursor (p) and the signal sequence-less form (m) of TorA-mCherry are indicated. (b) As in a, comparing crosslinking of the N-termini of TatA and TatB to wild-type TorA-mCherry (RR) and to its transport-incompetent mutant (KK). (c) As in a, using the three indicated N-terminal Bpa variants of TatA and TatB and the natural E. coli Tat substrate pSufI. In addition, the effects of dissipating the H+-motive force by the uncoupler CCCP on the crosslinking behaviour of pSufI to the N-termini of TatA and TatB are shown (red and green stars). The effectivity of CCCP is demonstrated by the failure of TatABC-INV (and others) to process the precursor of SufI (pSufI) to the signal sequence-less mature form (mSufI; lane 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Membrane-inserted RR-precursors touch at the N-termini of oligomeric TatB, subsequently also of TatA.(a) In vitro synthesis of the model RR-precursor TorA-mCherry in the presence of membrane vesicles (INV) containing wild-type TatABC, TatBC plus the G3Bpa variant of TatA and TatAC plus the F6Bpa variant of TatB. Crosslinking between these N-terminal Bpa variants and TorA-mCherry was initiated by ultraviolet light irradiation (+). Adducts of TorA-mCherry (TmC) to TatA (red stars) and TatB (green stars) could be purified via the His-tag of TorA-mCherry (His). The positions of the precursor (p) and the signal sequence-less form (m) of TorA-mCherry are indicated. (b) As in a, comparing crosslinking of the N-termini of TatA and TatB to wild-type TorA-mCherry (RR) and to its transport-incompetent mutant (KK). (c) As in a, using the three indicated N-terminal Bpa variants of TatA and TatB and the natural E. coli Tat substrate pSufI. In addition, the effects of dissipating the H+-motive force by the uncoupler CCCP on the crosslinking behaviour of pSufI to the N-termini of TatA and TatB are shown (red and green stars). The effectivity of CCCP is demonstrated by the failure of TatABC-INV (and others) to process the precursor of SufI (pSufI) to the signal sequence-less mature form (mSufI; lane 3).
Mentions: Next, we incubated INV that contained N-terminal Bpa variants of TatA or TatB with in vitro synthesized and radioactively labelled Tat substrates. In full agreement with a deep insertion, ultraviolet light-dependent adducts of TorA-mCherry to both N-termini of TatA and TatB (Fig. 4a; red and green stars) were observed. Notably, multiple adducts were obtained in either case. Because all of them were radioactively labelled and could be purified through the His-tag attached to TorA-mCherry (lanes 6 and 9), they all must contain the RR-precursor. Those multiple contacts are functionally relevant, because they were all abrogated when a transport-incompetent KK-mutant of TorA-mCherry was used (Fig. 4b). Very similar results were obtained for the natural E. coli Tat substrate pSufI (pFtsP) and three N-terminal Bpa variants of TatA and TatB each (Fig. 4c). These results clearly indicate that after insertion into the Tat translocase, RR-precursor molecules can associate simultaneously with the N-termini of multiple TatB and TatA molecules.

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