title: A mutation leading to super-assembly of twin-arginine translocase (Tat) protein complexes.
abstract: The Tat system transports folded proteins across the bacterial plasma membrane. The mechanism is believed to involve coalescence of a TatC-containing unit with a separate TatA complex, but the full translocation complex has never been visualised and the assembly process is poorly defined. We report the analysis of the Bacillus subtilis TatAyCy system, which occurs as separate TatAyCy and TatAy complexes at steady state, using single-particle electron microscopy (EM) and advanced atomic force microscopy (AFM) approaches. We show that a P2A mutation in the TatAy subunit leads to apparent super-assembly of Tat complexes. Purification of TatCy-containing complexes leads to a large increase in the TatA:TatC ratio, suggesting that TatAy(P2A) complexes may have attached to the TatAyCy complex. EM and AFM analyses show that the wild-type TatAyCy complex purifies as roughly spherical complexes of 9-16nm diameter, whereas the P2A mutation leads to accumulation of large (up to 500nm long) fibrils that are chains of numerous complexes. Time lapsed AFM imaging, recorded on fibrils under liquid, shows that they adopt a variety of tightly curved conformations, with radii of curvature of 10-12nm comparable to the size of single TatAy(P2A) complexes. The combined data indicate that the mutation leads to super-assembly of TatAy(P2A) complexes and we propose that an individual TatAy(P2A) complex assembles initially with a TatAy(P2A)Cy complex, after which further TatAy(P2A) complexes attach to each other. The data further suggest that the N-terminal extracytoplasmic domain of TatAy plays an essential role in Tat complex interactions.
authors.affiliation: School of Life Sciences, Gibbet Hill Campus, The University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Roshani
authors.initials: R
authors.lastname: Patel
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK.
authors.forename: Cvetelin
authors.initials: C
authors.lastname: Vasilev
authors.affiliation: School of Life Sciences, Gibbet Hill Campus, The University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Daniel
authors.initials: D
authors.lastname: Beck
authors.affiliation: Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Carmine G
authors.initials: CG
authors.lastname: Monteferrante
authors.affiliation: Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Jan Maarten
authors.initials: JM
authors.lastname: van Dijl
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK.
authors.forename: C Neil
authors.initials: CN
authors.lastname: Hunter
authors.affiliation: School of Life Sciences, Gibbet Hill Campus, The University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Corinne
authors.initials: C
authors.lastname: Smith
authors.affiliation: Centre for Molecular Processing, School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK. Electronic address: c.robinson-504@kent.ac.uk.
authors.forename: Colin
authors.initials: C
authors.lastname: Robinson
mesh_terms: D001120:Arginine; D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D002850:Chromatography, Gel; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D009154:Mutation / Q000235:genetics*; D017433:Protein Structure, Secondary
chemical_list: D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D046912:Multiprotein Complexes; D001120:Arginine
keywords: AFM; Atomic force microscopy; Signal peptide; Tat; Twin-arginine translocation