Cysteine scanning mutagenesis and topological mapping of the Escherichia coli twin-arginine translocase TatC component

TY - JOUR

T1 - Cysteine scanning mutagenesis and topological mapping of the Escherichia coli twin-arginine translocase TatC component

AU - Punginelli, Claire

AU - Maldonado, Barbara

AU - Grahl, Sabine

AU - Jack, Rachael

AU - Alami, Meriem

AU - Schroeder, Juliane

AU - Berks, Ben C.

AU - Palmer, Tracy

PY - 2007

Y1 - 2007

N2 - The TatC protein is an essential component of the Escherichia coli twin-arginine (Tat) protein translocation pathway. It is a polytopic membrane protein that forms a complex with TatB, together acting as the receptor for Tat substrates. In this study we have constructed 57 individual cysteine substitutions throughout the protein. Each of the substitutions resulted in a TatC protein that was competent to support Tat-dependent protein translocation. Accessibility studies with membrane-permeant and -impermeant thiol-reactive reagents demonstrated that TatC has six transmembrane helices, rather than the four suggested by a previous study (K. Gouffi, C.-L. Santini, and L.-F. Wu, FEBS Lett. 525:65-70, 2002). Disulfide cross-linking experiments with TatC proteins containing single cysteine residues showed that each transmembrane domain of TatC was able to interact with the same domain from a neighboring TatC protein. Surprisingly, only three of these cysteine variants retained the ability to cross-link at low temperatures. These results are consistent with the likelihood that most of the disulfide cross-links are-between TatC proteins in separate TatBC complexes, suggesting that TatC is located on the periphery of the complex.

AB - The TatC protein is an essential component of the Escherichia coli twin-arginine (Tat) protein translocation pathway. It is a polytopic membrane protein that forms a complex with TatB, together acting as the receptor for Tat substrates. In this study we have constructed 57 individual cysteine substitutions throughout the protein. Each of the substitutions resulted in a TatC protein that was competent to support Tat-dependent protein translocation. Accessibility studies with membrane-permeant and -impermeant thiol-reactive reagents demonstrated that TatC has six transmembrane helices, rather than the four suggested by a previous study (K. Gouffi, C.-L. Santini, and L.-F. Wu, FEBS Lett. 525:65-70, 2002). Disulfide cross-linking experiments with TatC proteins containing single cysteine residues showed that each transmembrane domain of TatC was able to interact with the same domain from a neighboring TatC protein. Surprisingly, only three of these cysteine variants retained the ability to cross-link at low temperatures. These results are consistent with the likelihood that most of the disulfide cross-links are-between TatC proteins in separate TatBC complexes, suggesting that TatC is located on the periphery of the complex.

U2 - 10.1128/JB.00647-07

DO - 10.1128/JB.00647-07

M3 - Article

SN - 0021-9193

VL - 189

SP - 5482

EP - 5494

JO - Journal of Bacteriology

JF - Journal of Bacteriology

IS - 15

ER -