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. 2010 Dec 1;21(23):4151–4161. doi: 10.1091/mbc.E10-07-0585

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© 2010 by The American Society for Cell Biology

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial –Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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Figure 4. — Incorporation of the cross-linker Bpa into TatB reveals multiple contacts between an extended part of the molecule and precursor proteins. (A) Model of TatB depicting its predicted transmembrane and amphipathic helices and the approximate positions of Bpa. (B) In vitro synthesis of radiolabeled pSufI in the presence of INVs containing the indicated Bpa variants of TatB. UV-irradiation yielded cross-links of varying intensities (asterisks) that were immunoprecipitated by anti-TatB antibodies. Note the different intensities by which the three positions on a helix turn of TatB cross-link to pSufI. (C) In vitro synthesis of radiolabeled pSufI in the presence of INVs containing the I36Bpa variant of TatB. Cross-linking was completely abolished if the signal sequence of pSufI carried a KK-mutation of the consensus RR-motif (lanes 1–6). Cross-linking was unimpaired (lanes 7–12), if INVs were added posttranslationally after depleting all energy sources to prevent translocation into INV (cf. Materials and Methods). Protease-protected SufI species shown in lane 7 demonstrate the translocation activity of the TatB(I36Bpa) variant and the lack of PK protection in lane 10 confirms the successful depletion of energy. (D) In vitro synthesis of radiolabeled pSufI in the presence of INVs containing the indicated Bpa variants of TatB. Note the adducts to TatB (L54Bpa) larger than 100 kDa that likely contain more than one TatB monomer. The origin of the TatB-adduct running at 90 kDa is not clear. This and the 75-kDa adduct were also obtained with a noncleavable mutant of pSufI (pSufI-ΔSP) indicating that all cross-links were between TatB and the precursor of SufI (lanes 15–19). Cross-linking of TatB to pSufI occurred independently of translocation (lanes 20–25). (E) In vitro synthesis of radiolabeled pSufI in the presence of INVs containing the indicated Bpa variants of TatB reveals a position-dependent cross-linking of the transmembrane helix of TatB to pSufI (lanes 1–12). Note the high molecular mass adduct to TatB(L9Bpa) indicative of a TatB oligomer. Cross-linking of TatB to pSufI occurred independently of translocation (lanes 13–18).