Sequence-specific Binding of prePhoD to Soluble TatA sub(d) Indicates Protein-Mediated Targeting of the Tat Export in Bacillus subtilis

• Published in: The Journal of biological chemistry 2003-10, Vol.278 (40), p.38428-38436
• Main Authors: Pop, OI, Westermann, M, Volkmer-Engert, R, Schulz, D, Lemke, C, Schreiber, S, Gerlach, R, Wetzker, R, Mueller, J P
• Format: Article
• Language: English
• Subjects: Bacillus subtilis; PhoD protein; phosphodiesterase; TatA protein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M306516200

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 (TatA sub(d)/TatC sub(d)) pair of proteins. Here, we show that TatA sub(d) was found beside its membrane-integrated localization in the cytosol were it interacted with prePhoD. prePhoD was efficiently co-immunoprecipitated by TatA sub(d). Inefficient co-immunoprecipitation of mature PhoD and missing interaction to Sec-dependent and cytosolic peptides by TatA sub(d) demonstrated a particular role of the twin-arginine signal peptide for this interaction. Affinity of prePhoD to TatA sub(d) was interfered by peptides containing the twin- arginine motif but remained active when the arginine residues were substituted. The selective binding of TatA sub(d) to peptides derived from the signal peptide of PhoD elucidated the function of the twin-arginine motif as a target site for pre-protein TatA sub(d) 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.