TAILIEUCHUNG - Báo cáo khoa học: Evidence for interactions between domains of TatA and TatB from mutagenesis of the TatABC subunits of the twin-arginine translocase

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial plasma membrane. Three subunits, TatA, B and C, are known to be involved but their modes of action are poorly understood, as are the inter-subunit interactions occurring within Tat complexes. We have generated mutations in the single transmembrane (TM) spans of TatA and TatB, with the aim of generating structural distortions. | ềFEBS Journal Evidence for interactions between domains of TatA and TatB from mutagenesis of the TatABC subunits of the twin-arginine translocase Claire M. L. Barrett and Colin Robinson Department of BiologicalSciences University of Warwick Coventry UK Keywords green fluorescent protein GFP Tat system twin-arginine protein transport signal peptide Correspondence C. Robinson Department of Biological Sciences University of Warwick Coventry CV4 7AL UK Fax 44 2476523701 Tel 44 2476523557 E-mail Crobinson@ Received 13 December 2004 revised 25 February 2005 accepted 8 March 2005 doi The twin-arginine translocation Tat system transports folded proteins across the bacterial plasma membrane. Three subunits TatA B and C are known to be involved but their modes of action are poorly understood as are the inter-subunit interactions occurring within Tat complexes. We have generated mutations in the single transmembrane TM spans of TatA and TatB with the aim of generating structural distortions. We show that substitution in TatB of three residues by glycine or a single residue by proline has no detectable effect on translocation whereas the presence of three glycines in the TatA TM span completely blocks Tat translocation activity. The results show that the integrity of the TatA TM span is vital for Tat activity whereas that of TatB can accommodate large-scale distortions. Near-complete restoration of activity in TatA mutants is achieved by the simultaneous presence of a V12P mutation in the TatB TM span strongly implying a direct functional interaction between the TatA B TM spans. We also analyzed the predicted amphipathic regions in TatA and TatB and again find evidence of direct interaction benign mutations in either subunit completely blocked translocation of two Tat substrates when present in combination. Finally we have re-examined the effects of previously analyzed TatABC mutations under conditions of high translocation .

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