PMID:17536842
| Citation |
Chan, CS, Zlomislic, MR, Tieleman, DP and Turner, RJ (2007) The TatA subunit of Escherichia coli twin-arginine translocase has an N-in topology. Biochemistry 46:7396-404 |
|---|---|
| Abstract |
The twin-arginine translocase (Tat) system is used by many bacteria to translocate folded proteins across the cytoplasmic membrane. The TatA subunit is the predicted pore-forming subunit and has been shown to form a homo-oligomeric complex. Through accessibility experiments using the thiol-reactive reagents 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid and Nalpha-(3-maleimidylproprionyl)biocytin toward site-specific cysteine mutants in TatA, we show that the N-terminus of TatA is located in the cytoplasm rather than the previously assumed periplasm. We also confirm previous observations that the C-terminus has a dual topology. By treatment with the membrane uncoupler carbonyl cyanide-m-chlorophenyl hydrazone, we show that the topological state of the C-terminus is dependent on the membrane potential. These results suggest two architectures of TatA in the membrane: one with a single transmembrane helix and the other with two transmembrane helices. Molecular models of both topologies were used to develop and cartoon a homo-oligomeric complex as a channel with a diameter of approximately 50 A and suggest that the double transmembrane helix topology might be the building block for the translocation channel. Additionally, in vivo cross-linking experiments of Gly2Cys and Thr22Cys mutants showed that Gly2, at the beginning of transmembrane helix-1, is in close proximity with Gly2 of a neighboring TatA, as Cys2 cross-linked immediately upon the addition of copper phenanthroline. On the other hand, Cys22, at the other end of the transmembrane helix, took at least 10 min to cross-link, suggesting that a possible movement or reorientation is required to bring this residue into proximity with a neighboring TatA subunit. |
| Links |
PubMed Online version:10.1021/bi7005288 |
| Keywords |
Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology; Cysteine/chemistry; Cysteine/genetics; Cytoplasm/chemistry; Cytoplasm/metabolism; Dimerization; Escherichia coli/enzymology; Escherichia coli/growth & development; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Histidine/chemistry; Membrane Potentials; Membrane Transport Proteins/chemistry; Membrane Transport Proteins/genetics; Models, Molecular; Point Mutation; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Subunits/chemistry; Protein Subunits/genetics; Protein Subunits/isolation & purification; Protein Subunits/metabolism; Uncoupling Agents/pharmacology |
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