A signal sequence suppressor mutant that stabilizes an assembled state of the twin arginine translocase

Abstract

The twin-arginine protein translocation (Tat) system mediates transport of folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. The Tat system of Escherichia coli is made up of TatA, TatB, and TatC components. TatBC comprise the substrate receptor complex, and active Tat translocases are formed by the substrate-induced association of TatA oligomers with this receptor. Proteins are targeted to TatBC by signal peptides containing an essential pair of arginine residues. We isolated substitutions, locating to the transmembrane helix of TatB that restored transport activity to Tat signal peptides with inactivating twin arginine substitutions. A subset of these variants also suppressed inactivating substitutions in the signal peptide binding site on TatC. The suppressors did not function by restoring detectable signal peptide binding to the TatBC complex. Instead, site-specific cross-linking experiments indicate that the suppressor substitutions induce conformational change in the complex and movement of the TatB subunit. The TatB F13Y substitution was associated with the strongest suppressing activity, even allowing transport of a Tat substrate lacking a signal peptide. In vivo analysis using a TatA-YFP fusion showed that the TatB F13Y substitution resulted in signal peptide-independent assembly of the Tat translocase. We conclude that Tat signal peptides play roles in substrate targeting and in triggering assembly of the active translocase.protein transport | twin arginine signal peptide | Tat pathway | genetic suppressor

The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding

“…In agreement with this, it has been observed that TatA-YFP was functionally inactive and activity could be only reestablished by co-production of non-tagged TatA or TatE (54). Substrate binding might influence the affinity of TatBC for TatA, as mutations that enhance the affinity of TatBC to Tat substrates show a constitutive TatA-YFP/TatBC interaction (55). An increased affinity of TatBC for TatA could therefore compensate for XFP-tag effects.…”

Signal peptide hydrophobicity modulates interaction with the twin-arginine translocase

“…It has been shown previously that a TatC F94D substitution inactivates Tat transport and that strains harboring this substitution are unable to grow on media containing the detergent SDS (27; Fig 1D). This phenotype arises due to an inability to export two Tat substrates, AmiA and AmiC that remodel the cell wall during growth (41, 42).…”

“…Since the signal peptide S12L substitution can act in trans to suppress inactivating substitutions in the TatC signal peptide binding site, we next asked whether it could act in cis to rescue inactivating substitutions at the twin-arginine motif. Previously it has been shown that substitutions of one or both consensus arginines of the SufI signal peptide are poorly tolerated (4), and indeed single substitutions of R6 to D, E, H, N or Q, or of R5R6 to KK, KH, KQ or HH in the SufIss-AmiA fusion are sufficient to prevent phenotypic growth of cells in the presence of SDS (27; Fig 3, Fig S1). Interestingly, however, introduction of the S12L substitution alongside any of the R6D, R6E, R6H, R6N, R6Q, or R5K/R6K restored strong growth of cells producing these fusion proteins in the presence of SDS (Fig 3A, Fig S1 panels B-G).…”

“…This phenotype arises due to an inability to export two Tat substrates, AmiA and AmiC that remodel the cell wall during growth (41, 42). We used a fusion protein whereby the signal peptide of SufI, which has the consensus F residue (Fig 1A) was fused to the mature region of AmiA (27) and constructed a random library of codon substitutions at F8. We then screened this library against a strain lacking native amiA / amiC and harboring TatC F94D , plating onto LB medium containing 2% SDS to select for suppressors of this inactivating substitution.…”

“…The signal peptide can also transition to a deep binding mode where it is inserted into the receptor complex, forming crosslinks to the transmembrane helix (TM) of TatB and TM5 of TatC (17, 19, 25, 26). Signal peptide insertion into the receptor drives structural reorganisation of the complex (13, 18, 26, 27) and the recruitment of further TatA molecules (19, 28-30). The assembled TatA oligomer mediates the transport of folded substrates across the membrane in an unknown manner, potentially by forming a translocation channel or by facilitating a localized weakening and transient disruption of the bilayer (31-33).…”