The Twin Arginine Consensus Motif of Tat Signal Peptides Is Involved in Sec-independent Protein Targeting in Escherichia coli

Abstract

In Escherichia coli a subset of periplasmic proteins is exported through the Tat pathway to which substrates are directed by an NH 2 -terminal signal peptide containing a consensus SRRXFLK "twin arginine" motif. The importance of the individual amino acids of the consensus motif for in vivo Tat transport has been assessed by site-directed mutagenesis of the signal peptide of the Tat substrate pre-SufI. Although the invariant arginine residues are crucial for efficient export, we find that slow transport of SufI is still possible if a single arginine is conservatively substituted by a lysine residue. Thus, in at least one signal peptide context there is no absolute dependence of Tat transport on the arginine pair. The consensus phenylalanine residue was found to be a critical determinant for efficient export but could be functionally substituted by leucine, another amino acid with a highly hydrophobic side chain. Unexpectedly, the consensus lysine residue was found to retard Tat transport. These observations and others suggest that the sequence conservation of the Tat consensus motif is a reflection of the functional importance of the consensus residues. Tat signal peptides characteristically have positively charged carboxyl-terminal regions. However, changing the sign of this charge does not affect export of SufI.

Functional analysis of a Campylobacter jejuni alkaline phosphatase secreted via the Tat export machinery

“…Analysis of the protein sequence of PhoA Cj indicated the presence of a typical twin-arginine (Tat) consensus motif in the N terminus. This RRxFLK motif, in which the twin-arginine is highly conserved (Stanley et al, 2000), led to the discovery that the C. jejuni phosphatase exploits the Tat secretion machinery to gain access to the periplasm. Evidence that the Tat system serves as transport machinery for PhoA Cj includes the lack of enzyme activity after substitution of the twin-arginine residues (Fig.…”

“…Analysis of the PhoA Cj protein revealed that it contains a conserved twinarginine domain near the N terminus, characteristic of proteins that are exported by the Tat secretion machinery (Stanley et al, 2000). To address whether PhoA Cj might be transported by the C. jejuni Tat system, we replaced the coding sequence for two arginine residues with two glycine residues in the phoA Cj located on plasmid pMA1-phoA Cj , containing the constitutively expressed metK promoter.…”

Structure of TatA Paralog, TatE, Suggests a Structurally Homogeneous Form of Tat Protein Translocase That Transports Folded Proteins of Differing Diameter

“…1 and 2). Tat substrates bear cleavable N-terminal signal peptides containing a critical and highly conserved twin-arginine motif within the consensus sequence (3,4). In Escherichia coli, the minimal set of components required for Tat-dependent translocation are three integral membrane proteins, TatA, TatB, and TatC, encoded by the tatABC operon, with molecular masses of 10, 18 and 30 kDa, respectively (5)(6)(7).…”

Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system

“…Proteins are targeted to the Tat pathway by N-terminal signal peptides containing a consensus amino acid sequence motif that includes two consecutive arginine residues (15)(16)(17). Tat-signal peptides are recognized at the membrane by a TatBC receptor complex (18)(19)(20)(21)(22)(23).…”