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J. Biol. Chem., Vol. 279, Issue 12, 11608-11615, March 19, 2004

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Dual Topology of the Escherichia coli TatA Protein^*

Kamila Gouffi, Fabien Gérard, Claire-Lise Santini, and Long-Fei Wu, Supported by Quality of Life and Management of Living Resources Grant QLK3-CT-1999-00917¶

From the Laboratoire de Chimie Bactérienne, UPR9043, Institut de Biologie Structurale et Microbiologie, CNRS, 31 chemin Joseph Aiguier, F-13402 Marseille cedex 20, France

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 -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.

Received for publication, December 3, 2003 , and in revised form, December 23, 2003.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address: Laboratoire de Biologie Moléculaire Eucaryote, UMR5099, CNRS, 118 Route de Narbonne, 31062 Toulouse Cedex 4 France.

Current address: Horticultural Sciences and Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL 32611.

^¶ To whom correspondence should be addressed. Tel.: 33-4-9116-4157; Fax: 33-4-9171-8914; E-mail: wu{at}ibsm.cnrs-mrs.fr.

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