PMID:14701831
| Citation |
Gouffi, K, Gérard, F, Santini, CL and Wu, LF (2004) Dual topology of the Escherichia coli TatA protein. J. Biol. Chem. 279:11608-15 |
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| Abstract |
The Escherichia coli Tat system has unusual capacity of translocating folded proteins across the cytoplasmic membrane. The TatA protein is the most abundant known Tat component and consists of a transmembrane segment followed by an amphipathic helix and a hydrophilic C terminus. To study the operation mechanism of the Tat apparatus, we analyzed the topology of TatA. Intriguingly, alkaline phosphatase (PhoA)-positive fusions were obtained at positions Gly-38, Lys-40, Asp-51, and Thr-53, which are all located at the cytoplasmic C terminus of the TatA protein. Interestingly, replacing phoA with uidA at Thr-53 led to positive beta-glucuronidase fusion, implying cytoplasmic location of the TatA C terminus. To further determine cellular localization of the TatA C terminus, we deleted the phoA gene and left 46 exogenous residues, including the tobacco etch virus (Tev) protease cleavage site (Tcs) after Thr-53, yielding TatA(T53)::Tcs. Unlike the PhoA and UidA fusions, which abolished the TatA function, the TatA(T53)::Tcs construct was able to restore the growth of tatA mutants on the minimal trimethlyamine N-oxide media. In vitro and in vivo proteolysis assay showed that the Tcs site of TatA(T53)::Tcs was accessible from both the periplasm and cytoplasm, indicating a dual topology of the TatA C terminus. Importantly, growth conditions seemed to influence the protein level of TatA and the cytoplasmic accessibility of the Tcs site of TatA(T53)::Tcs. A function-linked change of the TatA topology is suggested, and its implication in protein transport is discussed. |
| Links |
PubMed Online version:10.1074/jbc.M313187200 |
| Keywords |
Amino Acid Sequence; Base Sequence; DNA Primers; Escherichia coli/chemistry; Escherichia coli Proteins/chemistry; Molecular Sequence Data; Protein Structure, Secondary; Sequence Homology, Amino Acid |
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