Dual topology of the Escherichia coli TatA protein.
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.
Gouffi
Kamila
K
Laboratoire de Chimie Bactérienne, UPR9043, Institut de Biologie Structurale et Microbiologie, CNRS, 31 chemin Joseph Aiguier, F-13402 Marseille cedex 20, France.
Gérard
Fabien
F
Santini
Claire-Lise
CL
Wu
Long-Fei
LF
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D029968:Escherichia coli Proteins