ELUCIDATING THE QUALITY CONTROL MECHANISM OF THE TWIN-ARGININE TRANSLOCATION PATHWAY AND ITS EXPLOITATION FOR PROTEIN PRODUCTION

Abstract

The twin-arginine translocation (Tat) pathway involves an inherent quality control (QC) mechanism that assimilates proofreading of substrate protein folding with lipid bilayer export. However, the molecular attributes of how this QC mechanism is achieved remains poorly understood. Here, I hypothesized that the folding state of Tat substrates is “sensed” by the membrane-extrinsic domain of the TatB component of the Escherichia coli Tat translocase. In support of this hypothesis, removal of 80 membrane-extrinsic residues from the C-terminus of TatB led to the formation of functional translocases in vivo but with compromised quality control as corroborated by the uncharacteristic translocation of misfolded protein substrates. Furthermore, in vitro chaperone assays showed that the membrane- extrinsic domain of TatB possessed general chaperone activity, interacting with highly structured, early-unfolding intermediates of the model substrate protein, citrate synthase (CS). Collectively, my results shed light on how the Tat translocase may use chaperone-like client recognition to monitor the conformational status of its substrates. In addition to mechanistic insights that can be gained into the biology of Tat translocation, an understanding of how the Tat system exports substrates to the periplasm can be utilized for biotechnology purposes. E. coli remains one of the preferred hosts for biotech protein production due to its robust growth in culture and ease of genetic manipulation. Often it is desired to secrete these proteins into the periplasmic space over cytosolic production to decrease downstream purification costs and increase protein stability by preventing cytosolic proteolytic degradation. While it is beneficial to have the proteins in the periplasm, the bottleneck lies in the export of heterologous proteins across the membrane in sufficient quantities. To address this, I have engineered the Tat system for enhanced export of the antibody fragment, scFv13R4 (R4) into the periplasm using a directed co-evolution approach. Our selection yielded three unique hypersecretors of R4 with mutations in TatB and TatC. Further, the hypersecretors revealed quality control suppression alluding to a tight association between translocation and quality control, and that perhaps the quality control mechanism serves as a barrier retarding the kinetics of substrate translocation.