Evidence for interactions between domains of TatA and TatB from mutagenesis of the TatABC subunits of the twin‐arginine translocase

Abstract

The twin‐arginine translocation (Tat) system transports folded proteins across the bacterial plasma membrane. Three subunits, TatA, B and C, are known to be involved but their modes of action are poorly understood, as are the inter‐subunit interactions occurring within Tat complexes. We have generated mutations in the single transmembrane (TM) spans of TatA and TatB, with the aim of generating structural distortions. We show that substitution in TatB of three residues by glycine, or a single residue by proline, has no detectable effect on translocation, whereas the presence of three glycines in the TatA TM span completely blocks Tat translocation activity. The results show that the integrity of the TatA TM span is vital for Tat activity, whereas that of TatB can accommodate large‐scale distortions. Near‐complete restoration of activity in TatA mutants is achieved by the simultaneous presence of a V12P mutation in the TatB TM span, strongly implying a direct functional interaction between the TatA/B TM spans. We also analyzed the predicted amphipathic regions in TatA and TatB and again find evidence of direct interaction; benign mutations in either subunit completely blocked translocation of two Tat substrates when present in combination. Finally, we have re‐examined the effects of previously analyzed TatABC mutations under conditions of high translocation activity. Among numerous TatA or TatB mutations tested, TatA F39A alone blocked translocation, and only substitutions of P48 and F94 in TatC blocked translocation activity.

Salt Sensitivity of Minimal Twin Arginine Translocases

“…1). Several of these conserved residues had previously been shown to be important for TatA function in E. coli (43)(44)(45)(46)(47)(48)(49). Residues in TatAy were substituted with Ala, except for Pro-2, which was also replaced with Asp, and Ala-31, which was replaced with Gly.…”

“…The structure-function relationships in the TatA and TatB subunits of the TatABC translocase from E. coli have been studied in great detail (43)(44)(45)(46)(47)(48)(49). In contrast, relatively little is known about the bifunctional TatA subunits that are part of the TatAC translocases of Gram-positive bacteria.…”

Characterisation of the membrane-extrinsic domain of the TatB component of the twin arginine protein translocase

“…The amphipathic helix region of TatA is highly sensitive to inactivation by point mutation whereas substitutions in the same region of TatB had very little effect on TatB function [35,38,[40][41][42]. In this study we took an unbiased approach to isolate point mutations that resulted in a complete loss of TatB function.…”

Twin-arginine-dependent translocation of folded proteins

“…Furthermore, truncation of the C-terminal 40 amino acids of E. coli TatA was found not to negatively affect its activity [69]. An F39A mutation completely blocks Tat-dependent transport [61], but the mutant TatA assembles incorrectly in the membrane [70], whereas the K41A mutation destabilizes TatA [38]. Similarly important are residues F20 and G21 in the hinge region (figure 2b) [38,60] and Q8 at the proximal end of the TM helix, where a polar residue seems to be required for helix oligomerization [37].…”

“…P48A and F94A (figure 2c) were unanimously described as inactivating TatC [61,64,65,68], which in the case of P48A might be due to a destabilizing effect on the Tat(A)BC complex [70]. Y100A, Y126A and E170A substitutions show decreased activity [64].…”