The Transmembrane Glutamate Serves as a pH Sensor for Tha4 Oligomerization During Twin Arginine Transport of Proteins

TL;DR

A specific glutamate in Tha4 helps control protein transport by sensing pH and affecting how the protein assembles.

Contribution

The study reveals how transmembrane glutamate in Tha4 acts as a pH sensor and influences oligomerization during protein transport.

Findings

Alanine substitution of glutamate in Tha4 abolishes transport, while aspartate substitution partially restores it.

Tha4 oligomerization is influenced by precursor proteins and PMF, with alanine enhancing and aspartate reducing oligomerization.

The transmembrane glutamate is crucial for Tha4 activity and proper assembly during transport.

Abstract

Tha4, the smallest component of the cpTAT system, is thought to be the pore-forming element in the TAT translocase. A conserved glutamate at the 10th position in its transmembrane helix is crucial for function. Substitution of this glutamate with alanine abolishes transport, while aspartate substitution partially restores it, highlighting the importance of charge and hydrophobicity. To examine these effects, we generated Tha4 variants with different glutamate substitutions and assessed their transport abilities. Additionally, we developed assays to evaluate Tha4 oligomerization in the presence or absence of a proton motive force (PMF) and functional precursor proteins. Glutamate positional substitutions designed to increase proximity to the acidified lumen were not tolerated in the alanine background, whereas aspartate variants showed slight tolerance. Oligomerization assays revealed that Tha4 oligomer formation in the transmembrane helix region was primarily dependent on the presence of a functional precursor, regardless of PMF, while C-tail oligomer formation responded mainly to PMF. The amphipathic region showed no significant response to either factor. Alanine substitution enhanced oligomerization, while aspartate reduced it, likely due to altered packing interactions between monomers. These discoveries highlight the crucial function of the transmembrane glutamate in sustaining Tha4 activity and ensuring appropriate assembly during activation transport.

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