Abstract
The twin arginine protein transport (Tat) system translocates folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of chloroplasts. In Escherichia coli, TatA, TatB, and TatC are essential components of the machinery. A complex 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.
Highlights
The Tat pathway transports folded proteins across energy-coupling membranes
Our results clearly show that TatA has a fixed N-out, C-in topology that is not altered by the absence of other Tat components or by the overproduction of a Tat substrate
Overproduction of a Tat Substrate Does Not Alter the Topology of the TatA amphipathic helix (APH)—The results presented above support an N-out, C-in topology for TatA under all conditions tested and provide no evidence for dual topology of the APH suggested by others [28, 31]
Summary
The Tat pathway transports folded proteins across energy-coupling membranes. Results: TatA has a fixed N-out, C-in topology in intact cells that is not altered by the absence of other Tat components or overproduction of a Tat substrate. Some models for Tat transport assume that the APH of TatA may re-orient during transport (Fig. 1C), for example by folding into a channel assembled from TatA transmembrane helices like a trapdoor in response to a pulling force on the substrate [1, 29, 30]. Support for this model was provided by Gouffi et al [31], who used compartment-sensitive marker proteins fused to the end of the APH of TatA to infer that this region of TatA was exposed at both sides of the membrane. Our results clearly show that TatA has a fixed N-out, C-in topology that is not altered by the absence of other Tat components or by the overproduction of a Tat substrate
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