title: Sequence-specific binding of prePhoD to soluble TatAd indicates protein-mediated targeting of the Tat export in Bacillus subtilis.
abstract: The Tat (twin-arginine protein translocation) system initially discovered in the thylakoid membrane of chloroplasts has been described recently for a variety of eubacterial organisms. Although in Escherichia coli four Tat proteins with calculated membrane spanning domains have been demonstrated to mediate Tat-dependent transport, a specific transport system for twin-arginine signal peptide containing phosphodiesterase PhoD of Bacillus subtilis consists of one TatA/TatC (TatAd/TatCd) pair of proteins. Here, we show that TatAd was found beside its membrane-integrated localization in the cytosol were it interacted with prePhoD. prePhoD was efficiently co-immunoprecipitated by TatAd. Inefficient co-immunoprecipitation of mature PhoD and missing interaction to Sec-dependent and cytosolic peptides by TatAd demonstrated a particular role of the twin-arginine signal peptide for this interaction. Affinity of prePhoD to TatAd was interfered by peptides containing the twin-arginine motif but remained active when the arginine residues were substituted. The selective binding of TatAd to peptides derived from the signal peptide of PhoD elucidated the function of the twin-arginine motif as a target site for pre-protein TatAd interaction. Substitution of the binding motif demonstrated the pivotal role of basic amino acid residues for TatA binding. These features suggest that TatA interacts prior to membrane integration with its pre-protein substrate and could therefore assist targeting of twin-arginine pre-proteins.
authors.affiliation: Institut für Molekularbiologie, Friedrich-Schiller-Universität Jena, Hans-Knöll-Strasse 2, Jena D-07745, Germany.
authors.forename: Ovidiu I
authors.initials: OI
authors.lastname: Pop
authors.forename: Martin
authors.initials: M
authors.lastname: Westermann
authors.forename: Rudolf
authors.initials: R
authors.lastname: Volkmer-Engert
authors.forename: Daniela
authors.initials: D
authors.lastname: Schulz
authors.forename: Cornelius
authors.initials: C
authors.lastname: Lemke
authors.forename: Sandra
authors.initials: S
authors.lastname: Schreiber
authors.forename: Roman
authors.initials: R
authors.lastname: Gerlach
authors.forename: Reinhard
authors.initials: R
authors.lastname: Wetzker
authors.forename: Jörg P
authors.initials: JP
authors.lastname: Müller
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001412:Bacillus subtilis / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005614:Freeze Fracturing; D015696:Gene Products, tat / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D007150:Immunohistochemistry; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008854:Microscopy, Electron; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions
chemical_list: D029968:Escherichia coli Proteins; D015696:Gene Products, tat; D026901:Membrane Transport Proteins; D010455:Peptides; C499697:TatA protein, E coli; C461379:TatC protein, E coli; D006639:Histidine; D001120:Arginine; D010727:Phosphoric Diester Hydrolases