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 |
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creator | Barrett, Claire M.L. Mangels, Dorothea 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 |
doi_str_mv | 10.1016/j.jmb.2005.01.026 |
format | Article |
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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 <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. 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.</description><identifier>ISSN: 0022-2836</identifier><identifier>EISSN: 1089-8638</identifier><identifier>DOI: 10.1016/j.jmb.2005.01.026</identifier><identifier>PMID: 15740752</identifier><language>eng</language><publisher>England: Elsevier Ltd</publisher><subject>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</subject><ispartof>Journal of molecular biology, 2005-03, Vol.347 (2), p.453-463</ispartof><rights>2005 Elsevier Ltd</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c461t-a91fec4d95f919eff628c8b9105dd09ea58dede1e9c4000264b51589839d61d23</citedby><cites>FETCH-LOGICAL-c461t-a91fec4d95f919eff628c8b9105dd09ea58dede1e9c4000264b51589839d61d23</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.jmb.2005.01.026$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3548,27923,27924,45994</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15740752$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Barrett, Claire M.L.</creatorcontrib><creatorcontrib>Mangels, Dorothea</creatorcontrib><creatorcontrib>Robinson, Colin</creatorcontrib><title>Mutations in Subunits of the Escherichia coli Twin-arginine Translocase Block Function via Differing Effects on Translocation Activity or Tat Complex Structure</title><title>Journal of molecular biology</title><addtitle>J Mol Biol</addtitle><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 <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. 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.</description><subject>blue-native gel</subject><subject>Chromatography</subject><subject>Escherichia coli</subject><subject>Escherichia coli Proteins - chemistry</subject><subject>Escherichia coli Proteins - genetics</subject><subject>Escherichia coli Proteins - isolation & purification</subject><subject>Escherichia coli Proteins - metabolism</subject><subject>Membrane Transport Proteins - chemistry</subject><subject>Membrane Transport Proteins - genetics</subject><subject>Membrane Transport Proteins - isolation & purification</subject><subject>Membrane Transport Proteins - metabolism</subject><subject>Multiprotein Complexes</subject><subject>Mutation</subject><subject>Protein Structure, Quaternary</subject><subject>Protein Subunits - chemistry</subject><subject>Protein Subunits - genetics</subject><subject>Protein Subunits - isolation & purification</subject><subject>Protein Subunits - metabolism</subject><subject>protein transport</subject><subject>signal peptide</subject><subject>Tat system</subject><subject>twin-arginine</subject><issn>0022-2836</issn><issn>1089-8638</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkcFu1DAQhiMEotvCA3BBPnFLsJ3YscWpbLeAVMShy9ly7EnXS9ZZbGehT9NXxemu1Bs9zRy-_xtp_qJ4R3BFMOEft9V211UUY1ZhUmHKXxQLgoUsBa_Fy2KBMaUlFTU_K85j3OIM1o14XZwR1ja4ZXRRPHyfkk5u9BE5j26nbvIuRTT2KG0AraLZQHBm4zQy4-DQ-o_zpQ53zjsPaB20j8NodAT0Oc9f6HryZrahQ05cub7PaX-HVnkxs9Y_ZR6xy0wfXLpHY0BrndBy3O0H-ItuU5hMmgK8KV71eojw9jQvip_Xq_Xya3nz48u35eVNaRpOUqklyRcaK1kviYS-51QY0UmCmbVYgmbCggUC0jT5DZQ3HSNMSFFLy4ml9UXx4ejdh_H3BDGpnYsGhkF7GKeoeNu0jNf8WZDImmDO2PNgy2pO6GwkR9CEMcYAvdoHt9PhXhGs5p7VVuWe1dyzwkThx8z7k3zqdmCfEqdiM_DpCEB-2sFBUNE48AasC7kKZUf3H_0_NF-7HA</recordid><startdate>20050325</startdate><enddate>20050325</enddate><creator>Barrett, Claire M.L.</creator><creator>Mangels, Dorothea</creator><creator>Robinson, Colin</creator><general>Elsevier Ltd</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>20050325</creationdate><title>Mutations in Subunits of the Escherichia coli Twin-arginine Translocase Block Function via Differing Effects on Translocation Activity or Tat Complex Structure</title><author>Barrett, Claire M.L. ; Mangels, Dorothea ; Robinson, Colin</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c461t-a91fec4d95f919eff628c8b9105dd09ea58dede1e9c4000264b51589839d61d23</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>blue-native gel</topic><topic>Chromatography</topic><topic>Escherichia coli</topic><topic>Escherichia coli Proteins - chemistry</topic><topic>Escherichia coli Proteins - genetics</topic><topic>Escherichia coli Proteins - isolation & purification</topic><topic>Escherichia coli Proteins - metabolism</topic><topic>Membrane Transport Proteins - chemistry</topic><topic>Membrane Transport Proteins - genetics</topic><topic>Membrane Transport Proteins - isolation & purification</topic><topic>Membrane Transport Proteins - metabolism</topic><topic>Multiprotein Complexes</topic><topic>Mutation</topic><topic>Protein Structure, Quaternary</topic><topic>Protein Subunits - chemistry</topic><topic>Protein Subunits - genetics</topic><topic>Protein Subunits - isolation & 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. 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 <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. 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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