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Individual Subunits of the Eukaryotic Cytosolic Chaperonin Mediate Interactions with Binding Sites Located on Subdomains of β-Actin
The chaperonin containing TCP-1 (CCT) of eukaryotic cytosol is composed of eight different subunit species that are proposed to have independent functions in folding its in vivo substrates, the actins and tubulins. CCT has been loaded with 35S-β-actin by in vitro translation in reticulocyte lysate a...
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Published in: | The Journal of biological chemistry 2000-06, Vol.275 (25), p.18985-18994 |
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container_end_page | 18994 |
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container_title | The Journal of biological chemistry |
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creator | Hynes, Gillian M. Willison, Keith R. |
description | The chaperonin containing TCP-1 (CCT) of eukaryotic cytosol is composed of eight different subunit species that are proposed to have independent functions in folding its in vivo substrates, the actins and tubulins. CCT has been loaded with 35S-β-actin by in vitro translation in reticulocyte lysate and then subjected to immunoprecipitation with all eight anti-CCT subunit antibodies in mixed micelle buffers, conditions that disrupt CCT into its constituent monomers. Interactions between35S-β-actin and isolated CCTα, CCTβ, CCTε, or CCTθ subunits are observed, suggesting that polar and electrostatic interactions may mediate actin binding to these four CCT subunits. Additionally, a β-actin peptide array was screened for CCT-binding sequences. Three regions rich in charged and polar amino acid residues, which map to the surface of native β-actin, are implicated in interactions between actin and CCT. Several of these biochemical results are consistent with the recent cryo-electron microscopy three-dimensional structure of apo-CCT-α-actin, in which α-actin is bound by the apical domains of specific CCT subunits. A model is proposed in which actin interacts with several CCT subunits during its CCT-mediated folding cycle. |
doi_str_mv | 10.1074/jbc.M910297199 |
format | article |
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CCT has been loaded with 35S-β-actin by in vitro translation in reticulocyte lysate and then subjected to immunoprecipitation with all eight anti-CCT subunit antibodies in mixed micelle buffers, conditions that disrupt CCT into its constituent monomers. Interactions between35S-β-actin and isolated CCTα, CCTβ, CCTε, or CCTθ subunits are observed, suggesting that polar and electrostatic interactions may mediate actin binding to these four CCT subunits. Additionally, a β-actin peptide array was screened for CCT-binding sequences. Three regions rich in charged and polar amino acid residues, which map to the surface of native β-actin, are implicated in interactions between actin and CCT. Several of these biochemical results are consistent with the recent cryo-electron microscopy three-dimensional structure of apo-CCT-α-actin, in which α-actin is bound by the apical domains of specific CCT subunits. A model is proposed in which actin interacts with several CCT subunits during its CCT-mediated folding cycle.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1074/jbc.M910297199</identifier><identifier>PMID: 10748209</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Actins - chemistry ; Actins - metabolism ; Amino Acid Sequence ; Animals ; Binding Sites ; Chaperonin Containing TCP-1 ; Chaperonins - chemistry ; Chaperonins - genetics ; Chaperonins - metabolism ; Cytosol - metabolism ; Mice ; Models, Molecular ; Molecular Sequence Data ; Mutagenesis ; Protein Conformation</subject><ispartof>The Journal of biological chemistry, 2000-06, Vol.275 (25), p.18985-18994</ispartof><rights>2000 © 2000 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c425t-a1642cfa02cc11a1073b3eebac77fdb301cc15886ea09ae55c8fb457cda2c4df3</citedby><cites>FETCH-LOGICAL-c425t-a1642cfa02cc11a1073b3eebac77fdb301cc15886ea09ae55c8fb457cda2c4df3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S002192581980159X$$EHTML$$P50$$Gelsevier$$Hfree_for_read</linktohtml><link.rule.ids>314,780,784,3549,27924,27925,45780</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10748209$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hynes, Gillian M.</creatorcontrib><creatorcontrib>Willison, Keith R.</creatorcontrib><title>Individual Subunits of the Eukaryotic Cytosolic Chaperonin Mediate Interactions with Binding Sites Located on Subdomains of β-Actin</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>The chaperonin containing TCP-1 (CCT) of eukaryotic cytosol is composed of eight different subunit species that are proposed to have independent functions in folding its in vivo substrates, the actins and tubulins. CCT has been loaded with 35S-β-actin by in vitro translation in reticulocyte lysate and then subjected to immunoprecipitation with all eight anti-CCT subunit antibodies in mixed micelle buffers, conditions that disrupt CCT into its constituent monomers. Interactions between35S-β-actin and isolated CCTα, CCTβ, CCTε, or CCTθ subunits are observed, suggesting that polar and electrostatic interactions may mediate actin binding to these four CCT subunits. Additionally, a β-actin peptide array was screened for CCT-binding sequences. Three regions rich in charged and polar amino acid residues, which map to the surface of native β-actin, are implicated in interactions between actin and CCT. Several of these biochemical results are consistent with the recent cryo-electron microscopy three-dimensional structure of apo-CCT-α-actin, in which α-actin is bound by the apical domains of specific CCT subunits. A model is proposed in which actin interacts with several CCT subunits during its CCT-mediated folding cycle.</description><subject>Actins - chemistry</subject><subject>Actins - metabolism</subject><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Binding Sites</subject><subject>Chaperonin Containing TCP-1</subject><subject>Chaperonins - chemistry</subject><subject>Chaperonins - genetics</subject><subject>Chaperonins - metabolism</subject><subject>Cytosol - metabolism</subject><subject>Mice</subject><subject>Models, Molecular</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis</subject><subject>Protein Conformation</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2000</creationdate><recordtype>article</recordtype><recordid>eNp1kEFOwzAQRS0EoqWwZYl8gRTbSZp4WaoClVqxKEjsIseeUJfWrmKnqHtOxEE4Ew5Bgg2z8cj6_8_MQ-iSkiElWXK9LuVwwSlhPKOcH6E-JXkcxSl9PkZ9QhiNOEvzHjpzbk1CJZyeol5rzRnhffQ-M0rvtWrEBi-bsjHaO2wr7FeAp82rqA_Wa4knB2-d3bTdSuygtkYbvAClhQc8Mx5qIb22xuE37Vf4RodU84KX2oPDcyuDTGFr2hHKboU230M-P6JxsJlzdFKJjYOLn3eAnm6nj5P7aP5wN5uM55FMWOojQUcJk5UgTEpKRTgiLmOAUsgsq1QZExr-0zwfgSBcQJrKvCqTNJNKMJmoKh6gYZcra-tcDVWxq_U23FhQUrRMioCz-MUZDFedYdeUW1B_5B2_IMg7AYS19xrqwkkNRgYyNUhfKKv_y_4Cnb6H0A</recordid><startdate>20000623</startdate><enddate>20000623</enddate><creator>Hynes, Gillian M.</creator><creator>Willison, Keith R.</creator><general>Elsevier Inc</general><scope>6I.</scope><scope>AAFTH</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope></search><sort><creationdate>20000623</creationdate><title>Individual Subunits of the Eukaryotic Cytosolic Chaperonin Mediate Interactions with Binding Sites Located on Subdomains of β-Actin</title><author>Hynes, Gillian M. ; Willison, Keith R.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c425t-a1642cfa02cc11a1073b3eebac77fdb301cc15886ea09ae55c8fb457cda2c4df3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2000</creationdate><topic>Actins - chemistry</topic><topic>Actins - metabolism</topic><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Binding Sites</topic><topic>Chaperonin Containing TCP-1</topic><topic>Chaperonins - chemistry</topic><topic>Chaperonins - genetics</topic><topic>Chaperonins - metabolism</topic><topic>Cytosol - metabolism</topic><topic>Mice</topic><topic>Models, Molecular</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis</topic><topic>Protein Conformation</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hynes, Gillian M.</creatorcontrib><creatorcontrib>Willison, Keith R.</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hynes, Gillian M.</au><au>Willison, Keith R.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Individual Subunits of the Eukaryotic Cytosolic Chaperonin Mediate Interactions with Binding Sites Located on Subdomains of β-Actin</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>2000-06-23</date><risdate>2000</risdate><volume>275</volume><issue>25</issue><spage>18985</spage><epage>18994</epage><pages>18985-18994</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>The chaperonin containing TCP-1 (CCT) of eukaryotic cytosol is composed of eight different subunit species that are proposed to have independent functions in folding its in vivo substrates, the actins and tubulins. CCT has been loaded with 35S-β-actin by in vitro translation in reticulocyte lysate and then subjected to immunoprecipitation with all eight anti-CCT subunit antibodies in mixed micelle buffers, conditions that disrupt CCT into its constituent monomers. Interactions between35S-β-actin and isolated CCTα, CCTβ, CCTε, or CCTθ subunits are observed, suggesting that polar and electrostatic interactions may mediate actin binding to these four CCT subunits. Additionally, a β-actin peptide array was screened for CCT-binding sequences. Three regions rich in charged and polar amino acid residues, which map to the surface of native β-actin, are implicated in interactions between actin and CCT. Several of these biochemical results are consistent with the recent cryo-electron microscopy three-dimensional structure of apo-CCT-α-actin, in which α-actin is bound by the apical domains of specific CCT subunits. A model is proposed in which actin interacts with several CCT subunits during its CCT-mediated folding cycle.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>10748209</pmid><doi>10.1074/jbc.M910297199</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Actins - chemistry Actins - metabolism Amino Acid Sequence Animals Binding Sites Chaperonin Containing TCP-1 Chaperonins - chemistry Chaperonins - genetics Chaperonins - metabolism Cytosol - metabolism Mice Models, Molecular Molecular Sequence Data Mutagenesis Protein Conformation |
title | Individual Subunits of the Eukaryotic Cytosolic Chaperonin Mediate Interactions with Binding Sites Located on Subdomains of β-Actin |
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