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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

Formation of circular TatBC oligomers through specific contact sites of TatC.(a) Model of a TatBC tetramer looking down the membrane normal from the trans-side of the Tat translocase. Residues D63 and A133 of each TatC monomer are highlighted. (b) Membrane vesicles (INV) containing TatAB and either a Cys-less mutant of TatC (TatCΔCys) or the indicated single and double Cys mutants of TatC were either incubated with the sulfhydryl specific homobifunctional crosslinker BMH or mock treated. Proteins were dissolved in DTT-free SDS–PAGE loading buffer and analysed for the formation of TatC oligomers (blue stars) by SDS–PAGE and western blotting using anti-TatC antibodies.
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f7: Formation of circular TatBC oligomers through specific contact sites of TatC.(a) Model of a TatBC tetramer looking down the membrane normal from the trans-side of the Tat translocase. Residues D63 and A133 of each TatC monomer are highlighted. (b) Membrane vesicles (INV) containing TatAB and either a Cys-less mutant of TatC (TatCΔCys) or the indicated single and double Cys mutants of TatC were either incubated with the sulfhydryl specific homobifunctional crosslinker BMH or mock treated. Proteins were dissolved in DTT-free SDS–PAGE loading buffer and analysed for the formation of TatC oligomers (blue stars) by SDS–PAGE and western blotting using anti-TatC antibodies.

Mentions: On the basis of the two binding sites for TatB identified on the front side of a single TatC molecule (Fig. 2b,c) and the contacts of TatB monomers through their N-termini (Fig. 2d,e), it seems likely that the TatBC binding cavity for RR-precursors consists of a dome-like shell of TatB monomers that is surrounded by an outer shell made from TatC protomers. In Fig. 7a, we have modelled such a structure using four TatB and TatC molecules each and displayed it in top view looking down from the trans-side of the membrane. Each TatC monomer is approached by two TatB molecules, the TMs of which lie parallel to TM5 and cross TM2 and TM4 of TatC, respectively. In this model, the residues D63 and A133 of TatC (highlighted in yellow and orange), which crosslinking revealed as hotspots for tetramerization (Fig. 6a), would be positioned in some proximity. To verify this model, we replaced both residues either individually or jointly by Cys in an otherwise Cys-free variant of TatC24. We also constructed an M205C variant, because Bpa at this position of TatC caused much less tetramerization than D63 or A133 (Fig. 6a). INV containing those Cys variants of TatC were either incubated with the bifunctional Cys crosslinker Bismaleimidohexane (BMH) or mock treated and then probed by immuno-blotting against TatC antibodies for the formation of TatC complexes. Depending on the position, the single Cys mutants formed disulfides (Fig. 7b; lanes 1–8) reinforcing the dimerization tendency of TatC. If, however, INV contained the double Cys mutant D63C/A133C, larger adducts appeared that by size correspond to tetrameric and hexameric TatC complexes (lanes 9, 10). The formation of these oligomers was strongly favoured by BMH but was not obtained through direct oxidation by sodium tetrathionate (Supplementary Fig. 6), which is in line with a certain distance between D63 and A133 as proposed by the model in Fig. 7a. Because the tetramers and hexamers could not be obtained with the single Cys mutants, they must arise from mixed disulfides between D63 and A133. Two TatC molecules bridged through a single D63–A133 crosslink could then use their free D63C and A133C residues each to crosslink to a second dimer as illustrated in Fig. 7a. Inclusion of a third TatC dimer could then lead to a circular hexamer and so forth. Furthermore, the model places residue M205 at the tip of TM5 of TatC in a position that would be consistent with the weak oligomerization observed for the D63C/M205C double Cys mutant (Fig. 7b; lane 12).


Initial assembly steps of a translocase for folded proteins.

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

Formation of circular TatBC oligomers through specific contact sites of TatC.(a) Model of a TatBC tetramer looking down the membrane normal from the trans-side of the Tat translocase. Residues D63 and A133 of each TatC monomer are highlighted. (b) Membrane vesicles (INV) containing TatAB and either a Cys-less mutant of TatC (TatCΔCys) or the indicated single and double Cys mutants of TatC were either incubated with the sulfhydryl specific homobifunctional crosslinker BMH or mock treated. Proteins were dissolved in DTT-free SDS–PAGE loading buffer and analysed for the formation of TatC oligomers (blue stars) by SDS–PAGE and western blotting using anti-TatC antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Formation of circular TatBC oligomers through specific contact sites of TatC.(a) Model of a TatBC tetramer looking down the membrane normal from the trans-side of the Tat translocase. Residues D63 and A133 of each TatC monomer are highlighted. (b) Membrane vesicles (INV) containing TatAB and either a Cys-less mutant of TatC (TatCΔCys) or the indicated single and double Cys mutants of TatC were either incubated with the sulfhydryl specific homobifunctional crosslinker BMH or mock treated. Proteins were dissolved in DTT-free SDS–PAGE loading buffer and analysed for the formation of TatC oligomers (blue stars) by SDS–PAGE and western blotting using anti-TatC antibodies.
Mentions: On the basis of the two binding sites for TatB identified on the front side of a single TatC molecule (Fig. 2b,c) and the contacts of TatB monomers through their N-termini (Fig. 2d,e), it seems likely that the TatBC binding cavity for RR-precursors consists of a dome-like shell of TatB monomers that is surrounded by an outer shell made from TatC protomers. In Fig. 7a, we have modelled such a structure using four TatB and TatC molecules each and displayed it in top view looking down from the trans-side of the membrane. Each TatC monomer is approached by two TatB molecules, the TMs of which lie parallel to TM5 and cross TM2 and TM4 of TatC, respectively. In this model, the residues D63 and A133 of TatC (highlighted in yellow and orange), which crosslinking revealed as hotspots for tetramerization (Fig. 6a), would be positioned in some proximity. To verify this model, we replaced both residues either individually or jointly by Cys in an otherwise Cys-free variant of TatC24. We also constructed an M205C variant, because Bpa at this position of TatC caused much less tetramerization than D63 or A133 (Fig. 6a). INV containing those Cys variants of TatC were either incubated with the bifunctional Cys crosslinker Bismaleimidohexane (BMH) or mock treated and then probed by immuno-blotting against TatC antibodies for the formation of TatC complexes. Depending on the position, the single Cys mutants formed disulfides (Fig. 7b; lanes 1–8) reinforcing the dimerization tendency of TatC. If, however, INV contained the double Cys mutant D63C/A133C, larger adducts appeared that by size correspond to tetrameric and hexameric TatC complexes (lanes 9, 10). The formation of these oligomers was strongly favoured by BMH but was not obtained through direct oxidation by sodium tetrathionate (Supplementary Fig. 6), which is in line with a certain distance between D63 and A133 as proposed by the model in Fig. 7a. Because the tetramers and hexamers could not be obtained with the single Cys mutants, they must arise from mixed disulfides between D63 and A133. Two TatC molecules bridged through a single D63–A133 crosslink could then use their free D63C and A133C residues each to crosslink to a second dimer as illustrated in Fig. 7a. Inclusion of a third TatC dimer could then lead to a circular hexamer and so forth. Furthermore, the model places residue M205 at the tip of TM5 of TatC in a position that would be consistent with the weak oligomerization observed for the D63C/M205C double Cys mutant (Fig. 7b; lane 12).

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