Following the path of a twin-arginine precursor along the TatABC translocase of Escherichia coli.
The twin-arginine translocation (Tat) machinery present in bacterial and thylakoidal membranes is able to transport fully folded proteins. Consistent with previous in vivo data, we show that the model Tat substrate TorA-PhoA is translocated by the TatABC translocase of Escherichia coli inner membrane vesicles, only if the PhoA moiety was allowed to fold by disulfide bond formation. Although even unfolded TorA-PhoA was found to physically associate with the Tat translocase of the vesicles, site-specific cross-linking revealed a perturbed interaction of the signal sequence of unfolded TorA-PhoA with the TatBC receptor site. Some of the folded TorA-PhoA precursor accumulated in a partially protease-protected membrane environment, from where it could be translocated into the lumen of the vesicles upon re-installation of an H+-gradient. Translocation arrest occurred in immediate vicinity to TatA. Consistent with a neighborhood to TatA, TorA-PhoA remained protease-resistant in the presence of detergents that are known to preserve the oligomeric structures of TatA. Moreover, entry of TorA-PhoA to the protease-protected environment strictly required the presence of TatA. Collectively, our results are consistent with some degree of quality control by TatBC and a recruitment of TatA to a folded substrate that has functionally engaged the twin-arginine translocase.
Panahandeh
Sascha
S
Institut für Biochemie und Molekularbiologie, ZBMZ, Freiburg, Germany.
Maurer
Carlo
C
Moser
Michael
M
DeLisa
Matthew P
MP
Müller
Matthias
M
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D021381:Protein Transport / Q000502:physiology
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C408585:twin-arginine translocase complex, E coli; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase; D000469:Alkaline Phosphatase; C114089:phoA protein, E coli