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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 5. — TorAPhoA shows the same interaction with TatB as pSufI. (A) Surface-exposed residues I174, W270, I329, and L467 of PhoA used for the incorporation of the cross-linker Bpa. Structural data were taken from Wang et al., (2005). (B) The 55 kDa Tat precursor protein TorA-PhoA carrying Bpa at the surface position W270 (arrow) was synthesized in vitro with INVs added as indicated. When irradiated with UV-light, a major photo-cross-link (asterisk) was obtained that was recognized by anti-TatB antibodies (αTatB) and was absent from reactions devoid of membranes (lane 6). It was completely digested by Proteinase K (PK) (lane 3) in contrast to the PK-resistant translocated TorA-PhoA species (arrowheads) and therefore not membrane-protected. The three translocated species of TorA-PhoA from top to bottom are precursor, mature form and a translocation-arrested species (Panahandeh et al., 2008). Solubilization of the INVs' Tat machinery by 1% Triton X-100 prevented formation of the TatB adduct of TorA-PhoA (lane 5), whereas dissipation of the PMF by CCCP did not (lane 8). (C) TorA-PhoA (arrow) carrying Bpa at the surface position I329 was synthesized in vitro in the presence of INVs, irradiated with UV-light, and immunoprecipitated with antisera against TatA, TatB, and TatC (lanes 1–5). The only Tat-specific photo-adduct obtained was the one recognized by anti-TatB antibodies (asterisk). Cross-linking was completely abolished if the signal sequence of TorA-PhoA carried a KK-mutation of the consensus RR-motif (lanes 6–10). (D) Internal residues I99 and V196 of PhoA used for the incorporation of Bpa. (E) Comparison between the surface-exposed Bpa variant I329 of TorA-PhoA and the internal variant I99. Lanes 1–4 illustrate that transport of TorA-PhoA into INV proceeded only when TorA-PhoA was synthesized under oxidizing conditions, which were achieved by the addition of oxidized glutathione (GSSG). Transport is indicated in lane 4 by the appearance of the PK-resistant TorA-PhoA species specified in B (arrowheads). In contrast to the internal Bpa variant I99, the surface-exposed variant I329 formed adducts with TatB (asterisk) both under reducing and oxidizing conditions. (F) Cross-linking of the transmembrane and amphipathic helix of TatB to TorA-PhoA. The two TatB variants L9Bpa and L54Bpa again yielded higher molecular mass adducts (arrows). Cross-linking was observed both under reducing and oxidizing conditions.