Identification of key regions within the Escherichia coli TatAB subunits

Abstract

The twin-arginine translocation (Tat) system catalyzes the transport of folded proteins across the bacterial plasma membrane or the chloroplast thylakoid membrane. In Escherichia coli and most other species, three important tat genes have been identi¢ed but the structure and mechanism of this system are poorly understood; the role and location of TatA are particularly unclear. In this report we have used site-speci¢c mutagenesis to probe the signi¢cance of conserved features of the related TatA/B subunits. We ¢nd that an apparent 'hinge' region between the transmembrane (TM) span and an adjacent amphipathic region is important in both proteins, in that substitution of turn-inducing residues inhibits the export of a natural Tat substrate. Surprisingly, large-scale mutagenesis of the conserved amphipathic regions of TatA and TatB leads only to minor e¡ects on Tat-dependent export suggesting that this particular feature is not central to the translocation mechanism. This domain is, however, critical for the translocation process and we identify Gly/Pro residues in these regions of TatA/B that are essential for e⁄cient export. ß

Cysteine-scanning Mutagenesis and Disulfide Mapping Studies of the Conserved Domain of the Twin-arginine Translocase TatB Component

“…Strikingly, replacement of the highly conserved glutamate residue at position 8 with cysteine did not affect the measured Tat transport activity. This observation is consistent with previous reports that substitution of this residue in the E. coli protein with alanine was also well tolerated (48,49). The lowest periplasmic TMAO reductase activity was seen with the substitution of the isoleucine at residue 4.…”

“…Glu-8 is highly conserved in TatB sequences (5,57) and has been suggested to have a role in linking proton movements to the transport process (5,8). Surprisingly, substitution of Glu-8 in the E. coli TatB protein by either cysteine (this work), alanine (48,49), or glutamine (49) did not affect Tat transport activity, although a Glu to Gln substitution in the chloroplast TatB homolog affected association of the protein with TatC (58). These data suggest that Glu-8 may have a structural rather than mechanistic function in Tat transport as indicated by our modeling studies.…”

Dual Topology of the Escherichia coli TatA Protein

“…We observed that mutation in the hinge region affected the C terminus-out topology of TatA. Interestingly, it has been reported that the hinge region and the hydrophobicity of the amphipathic helix are crucial for the Tat function (17,18).…”

“…This finding suggests that the lysine 23 is essential for the membrane span topology of the TatA amphipathic helix. Interestingly, it has been recently reported that changing residues in the hinge region of TatA leads to the defect in TMAO reductase export (17,18).…”

“…C-terminal truncation analysis revealed that the transmembrane and amphipathic helical regions of TatA and TatB are critical for their function but that the C-terminal domains are not essential for Tat transport activity. Using site-specific mutagenesis to probe the significance of conserved features of the related TatA/B subunits leads to the conclusion that an apparent "hinge" region between the transmembrane segment and an APH is important in both proteins (17,18).…”