Volume 12, Issue 4, Pages 937-946 (October 2003) Differential Interactions between a Twin-Arginine Signal Peptide and Its Translocase in Escherichia coli  Meriem Alami, Iris Lüke, Sandra Deitermann, Gottfried Eisner, Hans-Georg Koch, Joseph Brunner, Matthias Müller  Molecular Cell  Volume 12, Issue 4, Pages 937-946 (October 2003) DOI: 10.1016/S1097-2765(03)00398-8

Figure 1 Incorporation of the Photoactivatable Crosslinker Tmd-Phe into preSufI (A) The signal sequence of preSufI is depicted with the consensus motif highlighted by a black box; the dashed line represents mature SufI. The positions are indicated where stop codons were introduced into the sufI DNA. Capital letters in front of the numerals indicate the amino acids which were replaced by Tmd-Phe. Numbering starts with the N-terminal methionine of the signal sequence of preSufI. (B) In vitro synthesis of preSufI by a coupled transcription/translation system prepared from E. coli. Expression of sufI DNA containing a TAG stop codon at position F8 is achieved by the addition of tRNAsup charged with Tmd-Phe. The asterisk marks a non-further-characterized translation product obtained in the absence of the suppressor tRNA. (C) Translocation of wild-type and mutant preSufI, which harbors Tmd-Phe at position L16, into cotranslationally added cytoplasmic membrane vesicles prepared from a TatABCD-overproducing E. coli strain (Tat+-INV). Transport is indicated by proteolytic processing of preSufI (white arrowhead) to mature SufI (closed arrowhead) and by proteinase K resistance (PK). Note the different nature of mature SufI, which is protease-resistant (closed arrowhead), and the unspecific translation product (*), which is not. Molecular Cell 2003 12, 937-946DOI: (10.1016/S1097-2765(03)00398-8)

Figure 2 Specific Interaction of TatA, TatB, and TatC with the RR Consensus Motif of preSufI under Varying Experimental Conditions (A) PreSufI carrying Tmd-Phe at position F8 was synthesized in vitro in 15-fold scaled-up reactions. Membrane vesicles were added posttranslationally. One reaction vol (50 μl) was precipitated directly with TCA (lanes 1 and 6) while the rest was irradiated with UV light. Thereafter again, 1 vol was precipitated directly with TCA (lanes 2 and 7), and 4 vol each was subjected to coimmunoprecipitation using antisera against TatA, TatB, and TatC. The position of preSufI on SDS-PAGE after phosphorimaging is indicated as well as the positions of marker proteins. X marks the TatA adduct. INV(Tat+) were obtained from E. coli strain BL21(DE3)pLysS overproducing TatABCD from plasmid pFAT65; INV(wt) were prepared from host strain BL21(DE3)pLysS expressing wild-type levels of TatABC. (B) Specificity controls for preSufI's interaction with the Tat proteins. Experimental conditions were as in (A). (C) Experimental conditions were as in (A). CCCP was added at a final concentration of 0.1 mM. (D) preSufI was synthesized in the presence of cotranslationally added Tat+-INV or low salt-extracted vesicles (wTat+-INV) depleted of the F1-ATPase. Molecular Cell 2003 12, 937-946DOI: (10.1016/S1097-2765(03)00398-8)

Figure 3 Upon Membrane Binding, preSufI Comes into Contact with TatB via Its Entire Signal Sequence and Even Downstream Regions (A) Precursors of SufI carrying Tmd-Phe as indicated were in vitro synthesized, incubated with wt-INV and Tat+-INV, and treated for UV-induced crosslinking and coimmunoprecipitation as described in Figure 2. (B) preSufI carrying Tmd-Phe at position L16 was incubated with Tat+-INV as in (A). CCCP was added at a final concentration of 0.1 mM. Molecular Cell 2003 12, 937-946DOI: (10.1016/S1097-2765(03)00398-8)

Figure 4 Molecular Contacts between preSufI and TatA, TatB, and TatC Reflect RR-Specific Recognition Events (A) UV-induced crosslinking of the indicated Tmd-Phe mutants of preSufI either containing a wild-type consensus motif (–RR–) or one in which the two arginines had been replaced by two lysines (–KK–). The KK mutations of the signal sequence are highlighted by two downward pointing arrows. Vesicles used were Tat+-INV. For further experimental details see Figure 2. (B) The indicated Tmd-Phe mutants of preSufI containing the wild-type RR consensus motif were allowed to bind to Tat+-INV in the presence of 0.4 mM of the wild signal peptide of SufI (RRsignal) or the signal peptide carrying the double KK mutation (KKsignal). Molecular Cell 2003 12, 937-946DOI: (10.1016/S1097-2765(03)00398-8)

Figure 5 Recognition of the RR Motif by TatC Is Independent of TatB, Whereas a Contact between the Signal Sequence of preSufI and TatB Is Abolished upon Removal or Overproduction of TatC (A) Crosslinking of preSufI via Tmd-Phe at position F8 to INV prepared from wild-type E. coli strain MC4100 (wt) or an isogenic TatB-deletion mutant BφD (ΔTatB). The crosslink to TatB is marked by an asterisk. For further details see legends to previous figures. (B) Immunoblot using antisera against TatC and TatB of membrane vesicles prepared from the indicated sources. wt, strain MC4100. Note the different level of TatC expression from plasmids p1TatC (pSDTc) and p2TatC (pFAT417). (C) Crosslinking of the indicated Tmd-Phe mutants of preSufI to INV derived from wild-type strain MC4100, a TatC-deletion mutant B1LKO (ΔTatC), and the ΔTatC strain transformed with the indicated TatC-expressing plasmids. Crosslinks to TatB are marked by asterisks. Molecular Cell 2003 12, 937-946DOI: (10.1016/S1097-2765(03)00398-8)

Figure 6 Contact between the Signal Sequence of preSufI and TatB Is Abolished upon Selective Overproduction of TatA and TatC (A) Crosslinking of the indicated Tmd-Phe mutants of preSufI to INV obtained from a strain expressing in addition to the chromosomal tat operon the TatA and TatC proteins from plasmid pFAT217 (INV-TatAC). (B) Immunoblot of INV obtained from the indicated sources. wt, host strain BL21(DE3)pLysS. Molecular Cell 2003 12, 937-946DOI: (10.1016/S1097-2765(03)00398-8)

Figure 7 No Evidence for the Occurrence of Ternary Complexes between preSufI, TatB, and TatC In vitro expression and UV-induced crosslinking to Tat+-INV of two preSufI constructs each simultaneously carrying two Tmd-Phe residues at the illustrated positions. Molecular Cell 2003 12, 937-946DOI: (10.1016/S1097-2765(03)00398-8)