The polar amino acid in the TatA transmembrane helix is not strictly necessary for protein function

The twin-arginine translocation (Tat) pathway utilizes the proton-motive force to transport folded proteins across cytoplasmic membranes in bacteria and archaea, as well as across the thylakoid membrane in plants and the inner membrane in mitochondria. In most species, the minimal components required for Tat activity consist of three subunits, TatA, TatB, and TatC. Previous studies have shown that a polar amino acid is present at the N terminus of the TatA transmembrane helix (TMH) across many different species. In order to systematically assess the functional importance of this polar amino acid in the TatA TMH in Escherichia coli, we examined a complete set of 19-amino-acid substitutions. Unexpectedly, although the polar amino acid is preferred overall, our experiments suggest that it is not necessary for a functional TatA. Hydrophilicity and helix-stabilizing properties of this polar amino acid were found to be highly correlated with the Tat activity. Specifically, change in charge status of the amino acid side chain due to pH resulted in a shift in hydrophilicity, which was demonstrated to impact the Tat transport activity. Furthermore, we identified a four-residue motif at the N terminus of the TatA TMH by sequence alignment. Using a biochemical approach, we found that the N-terminal motif was functionally significant, with evidence indicating a potential role in the preference for utilizing different proton-motive force components. Taken together, these findings yield new insights into the functionality of TatA and its potential role in the Tat transport mechanism.

Automated summary

The Tat pathway facilitates the transport of folded proteins across various membranes, and the polar amino acid at the N terminus of the TatA transmembrane helix was thought to be crucial. However, our experimental results in Escherichia coli showed that the polar amino acid is not necessary for TatA's functionality. Instead, the hydrophilicity and helix-stabilizing properties of this amino acid were found to strongly correlate with Tat activity. Additionally, we identified a four-residue motif at the N terminus of TatA's transmembrane helix, which may play a role in the preference for different proton-motive force components.