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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

Homo-oligomerization of TatC and its contacts to TatA are influenced by TatB.(a) Membrane vesicles containing the indicated Bpa variants of TatC (Fig. 2a) were probed for inter-TatC crosslinks (blue stars). Shown are western blots decorated with anti-TatC antibodies (αTatC). TatB–TatC adducts (black dots) had been identified as such in Fig. 2a and TatA–TatC adducts (squares) are addressed in b and c. (b,c) As in a, except that membrane vesicles used were devoid of TatB. Ultraviolet light-dependent adducts to the indicated Bpa variants of TatC were probed by antibodies against TatC (αTatC) and TatA (αTatA) revealing the 37 kDa TatA–TatC complexes (black squares). The same adduct was also obtained when Bpa was incorporated into the N terminus of TatA (c, lanes 4 and 6). TatA oligomers (red stars) crosslinked through the N terminus of TatA were also obtained. In addition, TatA forms dimers independently of Bpa (c, lanes 8–14, grey arrow head). Open squares mark an adduct that by size consists of two TatC and one TatA molecule. (d) Model of the E. coli TatC structure highlighting in yellow the three residues that yielded pronounced tetramers of TatC (a).
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f6: Homo-oligomerization of TatC and its contacts to TatA are influenced by TatB.(a) Membrane vesicles containing the indicated Bpa variants of TatC (Fig. 2a) were probed for inter-TatC crosslinks (blue stars). Shown are western blots decorated with anti-TatC antibodies (αTatC). TatB–TatC adducts (black dots) had been identified as such in Fig. 2a and TatA–TatC adducts (squares) are addressed in b and c. (b,c) As in a, except that membrane vesicles used were devoid of TatB. Ultraviolet light-dependent adducts to the indicated Bpa variants of TatC were probed by antibodies against TatC (αTatC) and TatA (αTatA) revealing the 37 kDa TatA–TatC complexes (black squares). The same adduct was also obtained when Bpa was incorporated into the N terminus of TatA (c, lanes 4 and 6). TatA oligomers (red stars) crosslinked through the N terminus of TatA were also obtained. In addition, TatA forms dimers independently of Bpa (c, lanes 8–14, grey arrow head). Open squares mark an adduct that by size consists of two TatC and one TatA molecule. (d) Model of the E. coli TatC structure highlighting in yellow the three residues that yielded pronounced tetramers of TatC (a).

Mentions: When the Bpa variants of TatC, which had been analysed for crosslinks to TatB (Fig. 2a), were screened for the occurrence of inter-TatC contacts, the results depicted in Fig. 6a were obtained. Besides the 50 kDa adducts (black dots), which due to their cross-reactivity with anti-TatB antibodies (Fig. 2a) represent 1:1 TatB–TatC complexes, additional 40 and 70 kDa adducts were obtained (blue stars) that were only recognized by the anti-TatC antibodies. The 40 kDa, most likely dimeric form of TatC was even observed for Bpa-free TatC when INV contained high amounts of TatC (Fig. 6a, lanes 3 and 4, blue star), similar to what has been observed previously3031. Otherwise, most of the Bpa variants of TatC shown in Fig. 6a dimerized upon activation by ultraviolet light (lanes 5–10 and 21–30, lower blue stars). A considerable amount of TatC63Bpa, however, prevailed as a 70 kDa adduct (lane 6, upper blue star), that is, most likely as a tetramer19. The same adduct was obtained for the 69Bpa and 133Bpa variants of TatC (lanes 8 and 14), indicating that distinct residues on the trans-side of TatC (Fig. 6d) seem to play an important role in mediating its oligomerization.


Initial assembly steps of a translocase for folded proteins.

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

Homo-oligomerization of TatC and its contacts to TatA are influenced by TatB.(a) Membrane vesicles containing the indicated Bpa variants of TatC (Fig. 2a) were probed for inter-TatC crosslinks (blue stars). Shown are western blots decorated with anti-TatC antibodies (αTatC). TatB–TatC adducts (black dots) had been identified as such in Fig. 2a and TatA–TatC adducts (squares) are addressed in b and c. (b,c) As in a, except that membrane vesicles used were devoid of TatB. Ultraviolet light-dependent adducts to the indicated Bpa variants of TatC were probed by antibodies against TatC (αTatC) and TatA (αTatA) revealing the 37 kDa TatA–TatC complexes (black squares). The same adduct was also obtained when Bpa was incorporated into the N terminus of TatA (c, lanes 4 and 6). TatA oligomers (red stars) crosslinked through the N terminus of TatA were also obtained. In addition, TatA forms dimers independently of Bpa (c, lanes 8–14, grey arrow head). Open squares mark an adduct that by size consists of two TatC and one TatA molecule. (d) Model of the E. coli TatC structure highlighting in yellow the three residues that yielded pronounced tetramers of TatC (a).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Homo-oligomerization of TatC and its contacts to TatA are influenced by TatB.(a) Membrane vesicles containing the indicated Bpa variants of TatC (Fig. 2a) were probed for inter-TatC crosslinks (blue stars). Shown are western blots decorated with anti-TatC antibodies (αTatC). TatB–TatC adducts (black dots) had been identified as such in Fig. 2a and TatA–TatC adducts (squares) are addressed in b and c. (b,c) As in a, except that membrane vesicles used were devoid of TatB. Ultraviolet light-dependent adducts to the indicated Bpa variants of TatC were probed by antibodies against TatC (αTatC) and TatA (αTatA) revealing the 37 kDa TatA–TatC complexes (black squares). The same adduct was also obtained when Bpa was incorporated into the N terminus of TatA (c, lanes 4 and 6). TatA oligomers (red stars) crosslinked through the N terminus of TatA were also obtained. In addition, TatA forms dimers independently of Bpa (c, lanes 8–14, grey arrow head). Open squares mark an adduct that by size consists of two TatC and one TatA molecule. (d) Model of the E. coli TatC structure highlighting in yellow the three residues that yielded pronounced tetramers of TatC (a).
Mentions: When the Bpa variants of TatC, which had been analysed for crosslinks to TatB (Fig. 2a), were screened for the occurrence of inter-TatC contacts, the results depicted in Fig. 6a were obtained. Besides the 50 kDa adducts (black dots), which due to their cross-reactivity with anti-TatB antibodies (Fig. 2a) represent 1:1 TatB–TatC complexes, additional 40 and 70 kDa adducts were obtained (blue stars) that were only recognized by the anti-TatC antibodies. The 40 kDa, most likely dimeric form of TatC was even observed for Bpa-free TatC when INV contained high amounts of TatC (Fig. 6a, lanes 3 and 4, blue star), similar to what has been observed previously3031. Otherwise, most of the Bpa variants of TatC shown in Fig. 6a dimerized upon activation by ultraviolet light (lanes 5–10 and 21–30, lower blue stars). A considerable amount of TatC63Bpa, however, prevailed as a 70 kDa adduct (lane 6, upper blue star), that is, most likely as a tetramer19. The same adduct was obtained for the 69Bpa and 133Bpa variants of TatC (lanes 8 and 14), indicating that distinct residues on the trans-side of TatC (Fig. 6d) seem to play an important role in mediating its oligomerization.

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