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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

Identification of mutual intramembrane contacts between TatB and TatC.(a) Membrane vesicles (INV) containing no Tat proteins (ΔTat), wild-type TatABC and TatAB together with the indicated Bpa variants of TatC (TatABCBpa) were irradiated with ultraviolet light (+) or mock treated (−) and then probed for TatB-cross-reactive adducts to TatC. Shown are western blots developed with anti-TatB antibodies (αTatB). Included are all of the TatCBpa variants shown in Fig. 1a in yellow, which yielded 1:1 complexes between TatB and TatC (black dot). (b,c) Models of E. coli TatC highlighting in orange residues that crosslinked to TatB according to a. The TatB-reactive sites cluster on two areas on the front of TatC invoking binding of two TatB monomers (TatB1, TatB2). The transmembrane helices (green) and the N-termini (red) of the two TatB monomers were modelled onto the TatC structure. (d) INV carrying the indicated Bpa variants in the N terminus of TatB were probed for TatB adducts on western blots using antibodies against TatB (αTatB). Indicated are predicted N-terminally colligated oligomers of TatB (green stars), as well as complexes between TatB and TatC (black dots and circles) and TatA (triangles). (e) As in d, using antibodies against TatC (αTatC). (f) As in d, using antibodies against TatA (αTatA).
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f2: Identification of mutual intramembrane contacts between TatB and TatC.(a) Membrane vesicles (INV) containing no Tat proteins (ΔTat), wild-type TatABC and TatAB together with the indicated Bpa variants of TatC (TatABCBpa) were irradiated with ultraviolet light (+) or mock treated (−) and then probed for TatB-cross-reactive adducts to TatC. Shown are western blots developed with anti-TatB antibodies (αTatB). Included are all of the TatCBpa variants shown in Fig. 1a in yellow, which yielded 1:1 complexes between TatB and TatC (black dot). (b,c) Models of E. coli TatC highlighting in orange residues that crosslinked to TatB according to a. The TatB-reactive sites cluster on two areas on the front of TatC invoking binding of two TatB monomers (TatB1, TatB2). The transmembrane helices (green) and the N-termini (red) of the two TatB monomers were modelled onto the TatC structure. (d) INV carrying the indicated Bpa variants in the N terminus of TatB were probed for TatB adducts on western blots using antibodies against TatB (αTatB). Indicated are predicted N-terminally colligated oligomers of TatB (green stars), as well as complexes between TatB and TatC (black dots and circles) and TatA (triangles). (e) As in d, using antibodies against TatC (αTatC). (f) As in d, using antibodies against TatA (αTatA).

Mentions: We next probed the membrane vesicles harbouring Bpa variants of TatC also for contacts between TatB and TatC. Crosslinking was induced by irradiating the INV with ultraviolet light, and the obtained adducts were analysed for the presence of TatB on western blots (Fig. 2a). Ultraviolet light-induced and TatB-cross-reactive adducts of ∼50 kDa, indicative of 1:1 TatBC complexes, appeared for residues located in the TM5 of TatC (202 and 205, black dot), consistent with the previously proposed alignment of the TM5 of TatC with the TM of TatB721. More prominent crosslinks of this size were, however, obtained for the Bpa157 variant and, as already previously recognized19, for Bpa150. As illustrated in Fig. 2b, these positions would not be in direct reach of the transmembrane helix of TatB lining up with the TM5 of TatC, but rather close to the more distally located amino (N) terminus of TatB, which according to recent NMR studies22 is depicted as a non-helical structure in Fig. 2b (in red).


Initial assembly steps of a translocase for folded proteins.

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

Identification of mutual intramembrane contacts between TatB and TatC.(a) Membrane vesicles (INV) containing no Tat proteins (ΔTat), wild-type TatABC and TatAB together with the indicated Bpa variants of TatC (TatABCBpa) were irradiated with ultraviolet light (+) or mock treated (−) and then probed for TatB-cross-reactive adducts to TatC. Shown are western blots developed with anti-TatB antibodies (αTatB). Included are all of the TatCBpa variants shown in Fig. 1a in yellow, which yielded 1:1 complexes between TatB and TatC (black dot). (b,c) Models of E. coli TatC highlighting in orange residues that crosslinked to TatB according to a. The TatB-reactive sites cluster on two areas on the front of TatC invoking binding of two TatB monomers (TatB1, TatB2). The transmembrane helices (green) and the N-termini (red) of the two TatB monomers were modelled onto the TatC structure. (d) INV carrying the indicated Bpa variants in the N terminus of TatB were probed for TatB adducts on western blots using antibodies against TatB (αTatB). Indicated are predicted N-terminally colligated oligomers of TatB (green stars), as well as complexes between TatB and TatC (black dots and circles) and TatA (triangles). (e) As in d, using antibodies against TatC (αTatC). (f) As in d, using antibodies against TatA (αTatA).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Identification of mutual intramembrane contacts between TatB and TatC.(a) Membrane vesicles (INV) containing no Tat proteins (ΔTat), wild-type TatABC and TatAB together with the indicated Bpa variants of TatC (TatABCBpa) were irradiated with ultraviolet light (+) or mock treated (−) and then probed for TatB-cross-reactive adducts to TatC. Shown are western blots developed with anti-TatB antibodies (αTatB). Included are all of the TatCBpa variants shown in Fig. 1a in yellow, which yielded 1:1 complexes between TatB and TatC (black dot). (b,c) Models of E. coli TatC highlighting in orange residues that crosslinked to TatB according to a. The TatB-reactive sites cluster on two areas on the front of TatC invoking binding of two TatB monomers (TatB1, TatB2). The transmembrane helices (green) and the N-termini (red) of the two TatB monomers were modelled onto the TatC structure. (d) INV carrying the indicated Bpa variants in the N terminus of TatB were probed for TatB adducts on western blots using antibodies against TatB (αTatB). Indicated are predicted N-terminally colligated oligomers of TatB (green stars), as well as complexes between TatB and TatC (black dots and circles) and TatA (triangles). (e) As in d, using antibodies against TatC (αTatC). (f) As in d, using antibodies against TatA (αTatA).
Mentions: We next probed the membrane vesicles harbouring Bpa variants of TatC also for contacts between TatB and TatC. Crosslinking was induced by irradiating the INV with ultraviolet light, and the obtained adducts were analysed for the presence of TatB on western blots (Fig. 2a). Ultraviolet light-induced and TatB-cross-reactive adducts of ∼50 kDa, indicative of 1:1 TatBC complexes, appeared for residues located in the TM5 of TatC (202 and 205, black dot), consistent with the previously proposed alignment of the TM5 of TatC with the TM of TatB721. More prominent crosslinks of this size were, however, obtained for the Bpa157 variant and, as already previously recognized19, for Bpa150. As illustrated in Fig. 2b, these positions would not be in direct reach of the transmembrane helix of TatB lining up with the TM5 of TatC, but rather close to the more distally located amino (N) terminus of TatB, which according to recent NMR studies22 is depicted as a non-helical structure in Fig. 2b (in red).

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