TY - JOUR

T1 - TatA and TatB generate a hydrophobic mismatch important for the function and assembly of the Tat translocon in Escherichia coli

AU - Mehner-Breitfeld, Denise

AU - Ringel, Michael Thomas

AU - Tichy, Daniel Alexander

AU - Endter, Laura J.

AU - Stroh, Kai Steffen

AU - Lünsdorf, Heinrich

AU - Risselada, Herr Jelger

AU - Brüser, Thomas

N1 - Funding Information: This study was supported by the DFG grant BR 2285/8-1 to T. B.

PY - 2022/9

Y1 - 2022/9

N2 - The twin-arginine translocation (Tat) system serves to translocate folded proteins across energy-transducing membranes in bacteria, archaea, plastids, and some mitochondria. In Escherichia coli, TatA, TatB, and TatC constitute functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) followed by an amphipathic helix. The TMHs of TatA and TatB generate a hydrophobic mismatch with the membrane, as the helices comprise only 12 consecutive hydrophobic residues; however, the purpose of this mismatch is unclear. Here, we shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both and analyzed effects on translocon function and assembly. We found the WT length helices functioned best, but some variation was clearly tolerated. Defects in function were exacerbated by simultaneous mutations in TatA and TatB, indicating partial compensation of mutations in each by the other. Furthermore, length variation in TatB destabilized TatBC-containing complexes, revealing that the 12-residue-length is important but not essential for this interaction and translocon assembly. To also address potential effects of helix length on TatA interactions, we characterized these interactions by molecular dynamics simulations, after having characterized the TatA assemblies by metal-tagging transmission electron microscopy. In these simulations, we found that interacting short TMHs of larger TatA assemblies were thinning the membrane and—together with laterally-aligned tilted amphipathic helices—generated a deep V-shaped membrane groove. We propose the 12 consecutive hydrophobic residues may thus serve to destabilize the membrane during Tat transport, and their conservation could represent a delicate compromise between functionality and minimization of proton leakage.

AB - The twin-arginine translocation (Tat) system serves to translocate folded proteins across energy-transducing membranes in bacteria, archaea, plastids, and some mitochondria. In Escherichia coli, TatA, TatB, and TatC constitute functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) followed by an amphipathic helix. The TMHs of TatA and TatB generate a hydrophobic mismatch with the membrane, as the helices comprise only 12 consecutive hydrophobic residues; however, the purpose of this mismatch is unclear. Here, we shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both and analyzed effects on translocon function and assembly. We found the WT length helices functioned best, but some variation was clearly tolerated. Defects in function were exacerbated by simultaneous mutations in TatA and TatB, indicating partial compensation of mutations in each by the other. Furthermore, length variation in TatB destabilized TatBC-containing complexes, revealing that the 12-residue-length is important but not essential for this interaction and translocon assembly. To also address potential effects of helix length on TatA interactions, we characterized these interactions by molecular dynamics simulations, after having characterized the TatA assemblies by metal-tagging transmission electron microscopy. In these simulations, we found that interacting short TMHs of larger TatA assemblies were thinning the membrane and—together with laterally-aligned tilted amphipathic helices—generated a deep V-shaped membrane groove. We propose the 12 consecutive hydrophobic residues may thus serve to destabilize the membrane during Tat transport, and their conservation could represent a delicate compromise between functionality and minimization of proton leakage.

KW - Tat Transport

KW - Proteintransport

KW - Tat transport

KW - Protein translocation

KW - computational biology

KW - membrane proteins

KW - stress response

KW - hydrophobic mismatch

KW - Tat system

KW - metal-tagging transmission electron microscopy (METTEM)

KW - membrane thinning

KW - protein translocation

UR - http://www.scopus.com/inward/record.url?scp=85136593028&partnerID=8YFLogxK

U2 - 10.1016/j.jbc.2022.102236

DO - 10.1016/j.jbc.2022.102236

M3 - Article

VL - 298

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 9

M1 - 102236

ER -