Escherichia coli TatA and TatB Proteins Have N-out, C-in Topology in Intact Cells

TY - JOUR

T1 - Escherichia coli TatA and TatB Proteins Have N-out, C-in Topology in Intact Cells

A1 - Koch,Sabrina

A1 - Fritsch,Maximilian J.

A1 - Buchanan,Grant

A1 - Palmer,Tracy

AU - Koch,Sabrina

AU - Fritsch,Maximilian J.

AU - Buchanan,Grant

AU - Palmer,Tracy

PY - 2012/4/27

Y1 - 2012/4/27

N2 - <p>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.</p>

AB - <p>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.</p>

U2 - 10.1074/jbc.M112.354555

DO - 10.1074/jbc.M112.354555

M1 - Article

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 18

VL - 287

SP - 14420

EP - 14431

ER -