Molecular dissection of TatC defines critical regions essential for protein transport and a TatB–TatC contact site

Abstract

The twin arginine transport (Tat) system transports folded proteins across the prokaryotic cytoplasmic membrane and the plant thylakoid membrane. TatC is the largest and most conserved component of the Tat machinery. It forms a multisubunit complex with TatB and binds the signal peptides of Tat substrates. Here we have taken a random mutagenesis approach to identify substitutions in Escherichia coli TatC that inactivate protein transport. We identify 32 individual amino acid substitutions that abolish or severely compromise TatC activity. The majority of the inactivating substitutions fall within the first two periplasmic loops of TatC. These regions are predicted to have conserved secondary structure and results of extensive amino acid insertion and deletion mutagenesis are consistent with these conserved elements being essential for TatC function. Three inactivating substitutions were identified in the fifth transmembrane helix of TatC. The inactive M205R variant could be suppressed by mutations affecting amino acids in the transmembrane helix of TatB. A physical interaction between TatC helix 5 and the TatB transmembrane helix was confirmed by the formation of a site-specific disulphide bond between TatC M205C and TatB L9C variants. This is the first molecular contact site mapped to single amino acid level between these two proteins.

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

“…Of these three, the F13Y substitution displayed the strongest suppressing activity, allowing export all of the twin arginine substitutions tested, and even allowing some translocation of AmiA completely devoid of a signal sequence. A combined bioinformatics and mutagenesis approach has shown the TatB transmembrane helix to bind along transmembrane helix 5 of TatC (50), and this is consistent with in vitro disulfide cross-linking studies (25,39). Signal peptide binding to the TatBC complex is suggested to cause movement of TatB from its resting-state binding site on TatC to a site elsewhere on the protein.…”

“…We introduced the individual amino acid substitutions F6Y, E8K, L9P, L9Q, L10P, F13Y, K30I, and I36N into TatB encoded within the tatABC operon on the very low copy number plasmid pTAT101 (39) and tested their ability to support export of wildtype AmiA and to suppress each of the RD, RE, RH, RN, RQ, KH, KQ, or HH AmiA signal sequence variants ( Fig. 1 B and C, Table 1, and SI Appendix, Fig.…”

“…Substrate-induced conformational changes in the wild-type complex can also be monitored by appearance of a cross-link between cysteine residues at position 205 at the periplasmic end of TatC transmembrane helix 5. This residue forms part of the TatB resting-state binding site (50), so is occluded from dimerization with a neighboring TatC molecule in the resting state, but has been shown to dimerize in response to overproduction of a Tat substrate (39). Similarly, assembly of TatA-YFP oligomers (indicating assembled translocation sites) that can be monitored by fluorescence microscopy is only seen for the wild-type translocase upon overproduction of Tat substrates (34).…”

Mapping the Signal Peptide Binding and Oligomer Contact Sites of the Core Subunit of the Pea Twin Arginine Protein Translocase

“…The E. coli mutation comparable to the L3 D276A was previously shown to disrupt TatC-TatB interactions (Buchanan et al, 2002). In addition, the homologous E. coli TM5 residues adjacent to TM5 mutations T272AP273A described here direct Cys-Cys cross-links with the TatB transmembrane domain (Kneuper et al, 2012;Rollauer et al, 2012). These thylakoid and E. coli TM5 residues are on the lumenal (periplasmic) end of TM5 close to L3 (P3).…”

“…Consistent with this general conclusion is the fact that the L3 residue Thr-275 directed strong cpTatC-cpTatC cross-linking (Figure 6), the E. coli TatC P3 residue Asp-211 directed photo-cross-linking to TatA (Tha4 ortholog) (Zoufaly et al, 2012), and the TM3-proximal P2 region residue 150 of E. coli TatC directed cross-linking to TatB and TatA (Zoufaly et al, 2012) Mutations in L1 resulted in complete loss of receptor complex and absence of any endogenous cpTatC in the purified mutant cpTatC ( Figure 5). The E. coli TatC periplasmic loop P1 (comparable to L1) is also hypersensitive to mutation (Kneuper et al, 2012) and at least one mutant in P1, P48A, resulted in loss of receptor complex (Barrett et al, 2005). Also of interest is that L1 Cys substitution directed strong cpTatC-cpTatC cross-linking (Figure 6), and a P1 residue of E. coli TatC directed photo-crosslinking to adjacent TatC (Zoufaly et al, 2012).…”