Mutations in Subunits of the Escherichia coli Twin-arginine Translocase Block Function via Differing Effects on Translocation Activity or Tat Complex Structure

We have used a combination of blue-native (BN) gel electrophoresis and protein purification to analyze the effects of TatA or TatC mutations on the structures of the primary TatABC and multimeric TatA complexes in Escherichia coli. Expression of wild-type TatABC leads to the production of a single m...

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Veröffentlicht in:Journal of molecular biology 2005-03, Vol.347 (2), p.453-463
Hauptverfasser: Barrett, Claire M.L., Mangels, Dorothea, Robinson, Colin
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Robinson, Colin
description We have used a combination of blue-native (BN) gel electrophoresis and protein purification to analyze the effects of TatA or TatC mutations on the structures of the primary TatABC and multimeric TatA complexes in Escherichia coli. Expression of wild-type TatABC leads to the production of a single major TatABC complex of 370 kDa and a heterogeneous set of TatA complexes of
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Expression of wild-type TatABC leads to the production of a single major TatABC complex of 370 kDa and a heterogeneous set of TatA complexes of &lt;100 kDa to ∼500 kDa. Two TatC mutations that block translocation have different effects on complex structures. P48A causes massive defects in TatABC assembly, including a marked separation of the TatBC subunits and the production of TatB and TatC aggregates. In contrast, TatABC complexes from the inactive TatC F94A mutant are structurally intact, suggesting that this mutation affects translocation activity rather than assembly. Neither TatC mutation affects the separate TatA complexes, showing that assembly of the TatA complexes is independent of TatABC assembly or activity. In contrast, three TatA mutations affect both the TatA and TatABC complexes. F39A assembles into smaller, incorrectly organized TatA complexes and the TatABC complexes contain an incorrect TatB:TatC ratio and unusually large amounts of TatA. A triple mutant in the amphipathic region forms slightly larger TatA complexes that are likewise disorganized, and a mutant containing three glycine substitutions in the transmembrane (TM) span assembles as grossly affected TatA complexes that are much larger than wild-type complexes. These mutants lead to a partial failure of TatB to assemble correctly. The data show that the amphipathic and TM regions play critical roles in TatA complex assembly. 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A triple mutant in the amphipathic region forms slightly larger TatA complexes that are likewise disorganized, and a mutant containing three glycine substitutions in the transmembrane (TM) span assembles as grossly affected TatA complexes that are much larger than wild-type complexes. These mutants lead to a partial failure of TatB to assemble correctly. The data show that the amphipathic and TM regions play critical roles in TatA complex assembly. 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purification</topic><topic>Protein Subunits - metabolism</topic><topic>protein transport</topic><topic>signal peptide</topic><topic>Tat system</topic><topic>twin-arginine</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Barrett, Claire M.L.</creatorcontrib><creatorcontrib>Mangels, Dorothea</creatorcontrib><creatorcontrib>Robinson, Colin</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of molecular biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Barrett, Claire M.L.</au><au>Mangels, Dorothea</au><au>Robinson, Colin</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Mutations in Subunits of the Escherichia coli Twin-arginine Translocase Block Function via Differing Effects on Translocation Activity or Tat Complex Structure</atitle><jtitle>Journal of molecular biology</jtitle><addtitle>J Mol Biol</addtitle><date>2005-03-25</date><risdate>2005</risdate><volume>347</volume><issue>2</issue><spage>453</spage><epage>463</epage><pages>453-463</pages><issn>0022-2836</issn><eissn>1089-8638</eissn><abstract>We have used a combination of blue-native (BN) gel electrophoresis and protein purification to analyze the effects of TatA or TatC mutations on the structures of the primary TatABC and multimeric TatA complexes in Escherichia coli. 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A triple mutant in the amphipathic region forms slightly larger TatA complexes that are likewise disorganized, and a mutant containing three glycine substitutions in the transmembrane (TM) span assembles as grossly affected TatA complexes that are much larger than wild-type complexes. These mutants lead to a partial failure of TatB to assemble correctly. The data show that the amphipathic and TM regions play critical roles in TatA complex assembly. All of the TatA mutations lead to partial or substantial defects in TatABC complex formation, demonstrating that the properties of TatA can have a marked influence on the TatABC complex.</abstract><cop>England</cop><pub>Elsevier Ltd</pub><pmid>15740752</pmid><doi>10.1016/j.jmb.2005.01.026</doi><tpages>11</tpages></addata></record>
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subjects blue-native gel
Chromatography
Escherichia coli
Escherichia coli Proteins - chemistry
Escherichia coli Proteins - genetics
Escherichia coli Proteins - isolation & purification
Escherichia coli Proteins - metabolism
Membrane Transport Proteins - chemistry
Membrane Transport Proteins - genetics
Membrane Transport Proteins - isolation & purification
Membrane Transport Proteins - metabolism
Multiprotein Complexes
Mutation
Protein Structure, Quaternary
Protein Subunits - chemistry
Protein Subunits - genetics
Protein Subunits - isolation & purification
Protein Subunits - metabolism
protein transport
signal peptide
Tat system
twin-arginine
title Mutations in Subunits of the Escherichia coli Twin-arginine Translocase Block Function via Differing Effects on Translocation Activity or Tat Complex Structure
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