Abstract
The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial membranes. Tat precursor proteins possess a conserved twin-arginine (RR) motif in their signal peptides that is involved in the binding of the proteins to the membrane-associated TatBC receptor complex. In addition, the hydrophobic region in the Tat signal peptides also contributes to TatBC binding, but whether regions beyond the signal-peptide cleavage site are involved in this process is unknown. Here, we analyzed the contribution of the early mature protein part of the Escherichia coli trimethylamine N-oxide reductase (TorA) to productive TatBC receptor binding. We identified substitutions in the 30 amino acids immediately following the TorA signal peptide (30aa-region) that restored export of a transport-defective TorA[KQ]-30aa-MalE precursor, in which the RR residues had been replaced by a lysine-glutamine pair. Some of these substitutions increased the hydrophobicity of the N-terminal part of the 30aa-region and thereby likely enhanced hydrophobic substrate-receptor interactions within the hydrophobic TatBC substrate-binding cavity. Another class of substitutions increased the positive net charge of the region's C-terminal part, presumably leading to strengthened electrostatic interactions between the mature substrate part and the cytoplasmic TatBC regions. Furthermore, we identified substitutions in the C-terminal domains of TatB following the transmembrane segment that restored transport of various transport-defective TorA-MalE derivatives. Some of these substitutions most likely affected the orientation or conformation of the flexible, carboxy-proximal helices of TatB. Therefore, we propose that a tight accommodation of the folded mature region by TatB contributes to productive binding of Tat substrates to TatBC.
Highlights
The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial membranes
The plasmid encoded TorA-MalE reporter protein consists of the signal peptide of the Tat substrate trimethylamine N-oxide reductase (TorA) and the first eight amino acid residues of the mature TorA protein fused to the mature protein part of the normally Sec-dependent maltose-binding protein (MalE) via a linker region consisting of the three amino acids Glu, Phe, and Asp
Because the presence of MalE in the periplasm is strictly required for maltose uptake [39], the Tat-specific export of TorA-MalE into the periplasm is directly linked with the ability of cells to utilize maltose and to grow on maltose minimal medium (MMM) as well as to form red colonies on MacConkey maltose (MCM) agar plates [23, 38, 40, 41]
Summary
The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial membranes. Tat precursor proteins possess a conserved twin-arginine (RR) motif in their signal peptides that is involved in the binding of the proteins to the membrane-associated TatBC receptor complex. We identified substitutions in the 30 amino acids immediately following the TorA signal peptide (30aa-region) that restored export of a transportdefective TorA[KQ]-30aa-MalE precursor, in which the RR residues had been replaced by a lysine– glutamine pair Some of these substitutions increased the hydrophobicity of the N-terminal part of the 30aa-region and thereby likely enhanced hydrophobic substrate–receptor interactions within the hydrophobic TatBC substrate-binding cavity. The signal peptide insertase activity of TatC promotes a deep, hairpin loop-like insertion of the signal peptide and most likely the early mature protein region into a membrane-embedded, enclosed binding cavity formed by both subunits of the substrate receptor [29], a process that was commonly thought to strictly depend on an intact RR motif [13]. Recent studies showed that Tat signal peptides lacking the highly conserved arginine residues in the Tat consensus motif can be threaded deep into the TatBC-binding cav-
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
