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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

RR-signal peptides are sequestered within a TatBC-made insertion cavity.(a) N-terminal sequence of the natural RR-precursor pSufI highlighting the residues that were exchanged against Cys. The six amino acids flanking the cleavage site (slash) of the signal peptide are underlined. (b) Relative labelling by mal-PEG of the indicated Cys mutants of pSufI following their in vitro synthesis and incubation with ΔTat-INV, that is, under conditions, in which they could not interact with the Tat translocase. Indicated are the mean ratios between labelled and unlabelled pSufI species obtained from two or three parallel experiments shown in c (lane ΔTat). Where an error bar is shown, the data represent the mean ± standard deviation of three parallel experiments. Otherwise, the mean of two experiments is shown. Supporting data from each individual experiment are shown in Supplementary Table 1. About 50–60% of each Cys mutant became thus labelled with mal-PEG, except for P22 (for unknown reasons) and residues in the early mature region of SufI (due to folding). (c) The Cys variants of pSufI and one transport-incompetent KK-derivative, which are specified on the left-hand side, were synthesized and radiolabelled in vitro, incubated with INV containing the indicated Tat proteins, and reacted with mal-PEG. Shown are the PEGylated (white arrowhead) and unmodified (black arrowhead) species of pSufI following SDS–PAGE and phosphorimaging. (d) Quantitative data obtained from two or three parallel experiments shown in c. Values were normalized to the labelling efficiency obtained in the presence of ΔTat-INV. Where an error bar is shown, the data represent the mean ± standard deviation of three parallel experiments. Otherwise, the mean of two experiments is shown. Supporting data from each individual experiment are shown in Supplementary Table 2. Protection against PEGylation was obtained for positions within the signal sequence of pSufI but not for flanking residues. Protection by INV was more pronounced in the presence of TatB than in its absence.
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f5: RR-signal peptides are sequestered within a TatBC-made insertion cavity.(a) N-terminal sequence of the natural RR-precursor pSufI highlighting the residues that were exchanged against Cys. The six amino acids flanking the cleavage site (slash) of the signal peptide are underlined. (b) Relative labelling by mal-PEG of the indicated Cys mutants of pSufI following their in vitro synthesis and incubation with ΔTat-INV, that is, under conditions, in which they could not interact with the Tat translocase. Indicated are the mean ratios between labelled and unlabelled pSufI species obtained from two or three parallel experiments shown in c (lane ΔTat). Where an error bar is shown, the data represent the mean ± standard deviation of three parallel experiments. Otherwise, the mean of two experiments is shown. Supporting data from each individual experiment are shown in Supplementary Table 1. About 50–60% of each Cys mutant became thus labelled with mal-PEG, except for P22 (for unknown reasons) and residues in the early mature region of SufI (due to folding). (c) The Cys variants of pSufI and one transport-incompetent KK-derivative, which are specified on the left-hand side, were synthesized and radiolabelled in vitro, incubated with INV containing the indicated Tat proteins, and reacted with mal-PEG. Shown are the PEGylated (white arrowhead) and unmodified (black arrowhead) species of pSufI following SDS–PAGE and phosphorimaging. (d) Quantitative data obtained from two or three parallel experiments shown in c. Values were normalized to the labelling efficiency obtained in the presence of ΔTat-INV. Where an error bar is shown, the data represent the mean ± standard deviation of three parallel experiments. Otherwise, the mean of two experiments is shown. Supporting data from each individual experiment are shown in Supplementary Table 2. Protection against PEGylation was obtained for positions within the signal sequence of pSufI but not for flanking residues. Protection by INV was more pronounced in the presence of TatB than in its absence.

Mentions: To provide independent experimental evidence for the sequestration of an RR-signal peptide within an intramembrane insertion cavity, we probed for its accessibility towards a branched form of maleinimido-polyethylene glycol (mal-PEG). To this end, single Cys mutants of pSufI (Fig. 5a) were synthesized in vitro, subsequently incubated with INV containing various combinations of the TatABC subunits, labelled with mal-PEG and analysed by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and phosphorimaging. The experimental conditions were adjusted so as to largely prevent membrane permeation of mal-PEG and removal of pSufI from TatABC due to transport in, and cleavage by, the membrane vesicles. In this way, 50–60% of the molecules of most Cys-bearing pSufI mutants became labelled with mal-PEG, when they were incubated with INV that lacked the TatABC proteins (Fig. 5b; Supplementary Table 1). Labelling of pSufI-C22 was markedly impaired for unknown reasons, whereas the reduced accessibility of most residues beyond position 30 was due to their sequestration within the folded part of pSufI, as verified by an increase in PEGylation of residue V36 in the presence of 8 M urea and the unrestricted labelling of the surface-exposed29 residue W441 (Supplementary Fig. 4).


Initial assembly steps of a translocase for folded proteins.

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

RR-signal peptides are sequestered within a TatBC-made insertion cavity.(a) N-terminal sequence of the natural RR-precursor pSufI highlighting the residues that were exchanged against Cys. The six amino acids flanking the cleavage site (slash) of the signal peptide are underlined. (b) Relative labelling by mal-PEG of the indicated Cys mutants of pSufI following their in vitro synthesis and incubation with ΔTat-INV, that is, under conditions, in which they could not interact with the Tat translocase. Indicated are the mean ratios between labelled and unlabelled pSufI species obtained from two or three parallel experiments shown in c (lane ΔTat). Where an error bar is shown, the data represent the mean ± standard deviation of three parallel experiments. Otherwise, the mean of two experiments is shown. Supporting data from each individual experiment are shown in Supplementary Table 1. About 50–60% of each Cys mutant became thus labelled with mal-PEG, except for P22 (for unknown reasons) and residues in the early mature region of SufI (due to folding). (c) The Cys variants of pSufI and one transport-incompetent KK-derivative, which are specified on the left-hand side, were synthesized and radiolabelled in vitro, incubated with INV containing the indicated Tat proteins, and reacted with mal-PEG. Shown are the PEGylated (white arrowhead) and unmodified (black arrowhead) species of pSufI following SDS–PAGE and phosphorimaging. (d) Quantitative data obtained from two or three parallel experiments shown in c. Values were normalized to the labelling efficiency obtained in the presence of ΔTat-INV. Where an error bar is shown, the data represent the mean ± standard deviation of three parallel experiments. Otherwise, the mean of two experiments is shown. Supporting data from each individual experiment are shown in Supplementary Table 2. Protection against PEGylation was obtained for positions within the signal sequence of pSufI but not for flanking residues. Protection by INV was more pronounced in the presence of TatB than in its absence.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: RR-signal peptides are sequestered within a TatBC-made insertion cavity.(a) N-terminal sequence of the natural RR-precursor pSufI highlighting the residues that were exchanged against Cys. The six amino acids flanking the cleavage site (slash) of the signal peptide are underlined. (b) Relative labelling by mal-PEG of the indicated Cys mutants of pSufI following their in vitro synthesis and incubation with ΔTat-INV, that is, under conditions, in which they could not interact with the Tat translocase. Indicated are the mean ratios between labelled and unlabelled pSufI species obtained from two or three parallel experiments shown in c (lane ΔTat). Where an error bar is shown, the data represent the mean ± standard deviation of three parallel experiments. Otherwise, the mean of two experiments is shown. Supporting data from each individual experiment are shown in Supplementary Table 1. About 50–60% of each Cys mutant became thus labelled with mal-PEG, except for P22 (for unknown reasons) and residues in the early mature region of SufI (due to folding). (c) The Cys variants of pSufI and one transport-incompetent KK-derivative, which are specified on the left-hand side, were synthesized and radiolabelled in vitro, incubated with INV containing the indicated Tat proteins, and reacted with mal-PEG. Shown are the PEGylated (white arrowhead) and unmodified (black arrowhead) species of pSufI following SDS–PAGE and phosphorimaging. (d) Quantitative data obtained from two or three parallel experiments shown in c. Values were normalized to the labelling efficiency obtained in the presence of ΔTat-INV. Where an error bar is shown, the data represent the mean ± standard deviation of three parallel experiments. Otherwise, the mean of two experiments is shown. Supporting data from each individual experiment are shown in Supplementary Table 2. Protection against PEGylation was obtained for positions within the signal sequence of pSufI but not for flanking residues. Protection by INV was more pronounced in the presence of TatB than in its absence.
Mentions: To provide independent experimental evidence for the sequestration of an RR-signal peptide within an intramembrane insertion cavity, we probed for its accessibility towards a branched form of maleinimido-polyethylene glycol (mal-PEG). To this end, single Cys mutants of pSufI (Fig. 5a) were synthesized in vitro, subsequently incubated with INV containing various combinations of the TatABC subunits, labelled with mal-PEG and analysed by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and phosphorimaging. The experimental conditions were adjusted so as to largely prevent membrane permeation of mal-PEG and removal of pSufI from TatABC due to transport in, and cleavage by, the membrane vesicles. In this way, 50–60% of the molecules of most Cys-bearing pSufI mutants became labelled with mal-PEG, when they were incubated with INV that lacked the TatABC proteins (Fig. 5b; Supplementary Table 1). Labelling of pSufI-C22 was markedly impaired for unknown reasons, whereas the reduced accessibility of most residues beyond position 30 was due to their sequestration within the folded part of pSufI, as verified by an increase in PEGylation of residue V36 in the presence of 8 M urea and the unrestricted labelling of the surface-exposed29 residue W441 (Supplementary Fig. 4).

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