TY - JOUR
T1 - Escherichia coli TatA and TatB Proteins Have N-out, C-in Topology in Intact Cells
AU - Koch, Sabrina
AU - Fritsch, Maximilian J.
AU - Buchanan, Grant
AU - Palmer, Tracy
PY - 2012/4/27
Y1 - 2012/4/27
N2 - The twin arginine protein transport (Tat) system translocates folded proteins across the cytoplasmic membrane of pro-karyotes and the thylakoid membrane of chloroplasts. In Escherichia coli, TatA, TatB, and TatC are essential components of the machinery. Acomplex of TatB and TatC acts as the substrate receptor, whereas TatA is proposed to form the Tat transport channel. TatA and TatB are related proteins that comprise an N-terminal transmembrane helix and an adjacent amphipathic helix. Previous studies addressing the topological organization of TatA have given conflicting results. In this study, we have addressed the topological arrangement of TatA and TatB in intact cells by labeling of engineered cysteine residues with the membrane-impermeable thiol reagent methoxypolyethylene glycol maleimide. Our results show that TatA and TatB share an N-out, C-in topology, with no evidence that the amphipathic helices of either protein are exposed at the periplasmic side of the membrane. We further show that the N-out, C-in topology of TatA is fixed and is not affected by the absence of other Tat components or by the overproduction of a Tat substrate. These data indicate that topological reorganization of TatA is unlikely to accompany Tat-dependent protein transport.
AB - The twin arginine protein transport (Tat) system translocates folded proteins across the cytoplasmic membrane of pro-karyotes and the thylakoid membrane of chloroplasts. In Escherichia coli, TatA, TatB, and TatC are essential components of the machinery. Acomplex of TatB and TatC acts as the substrate receptor, whereas TatA is proposed to form the Tat transport channel. TatA and TatB are related proteins that comprise an N-terminal transmembrane helix and an adjacent amphipathic helix. Previous studies addressing the topological organization of TatA have given conflicting results. In this study, we have addressed the topological arrangement of TatA and TatB in intact cells by labeling of engineered cysteine residues with the membrane-impermeable thiol reagent methoxypolyethylene glycol maleimide. Our results show that TatA and TatB share an N-out, C-in topology, with no evidence that the amphipathic helices of either protein are exposed at the periplasmic side of the membrane. We further show that the N-out, C-in topology of TatA is fixed and is not affected by the absence of other Tat components or by the overproduction of a Tat substrate. These data indicate that topological reorganization of TatA is unlikely to accompany Tat-dependent protein transport.
UR - http://www.scopus.com/inward/record.url?scp=84860354294&partnerID=8YFLogxK
U2 - 10.1074/jbc.M112.354555
DO - 10.1074/jbc.M112.354555
M3 - Article
C2 - 22399293
SN - 0021-9258
VL - 287
SP - 14420
EP - 14431
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 18
ER -