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PMID:16762366

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Citation

Pappenberger, G, McCormack, EA and Willison, KR (2006) Quantitative actin folding reactions using yeast CCT purified via an internal tag in the CCT3/gamma subunit. J. Mol. Biol. 360:484-96

Abstract

The eukaryotic cytosolic chaperonin CCT is an essential ATP-dependent protein folding machine whose action is required for folding the cytoskeletal proteins actin and tubulin, and a small number of other substrates, including members of the WD40-propellor repeat-containing protein family. An efficient purification protocol for CCT from Saccharomyces cerevisiae has been developed. It uses the calmodulin binding peptide as an affinity tag in an internal loop in the apical domain of the CCT3 subunit, which is predicted to be located on the outside of the double-ring assembly. This purified yeast CCT was used for a novel quantitative actin-folding assay with human beta-actin or yeast ACT1p protein folding intermediates, Ac(I), pre-synthesised in an Escherichia coli translation system. The formation of native actin follows approximately a first-order reaction with a rate constant of about 0.03 min(-1). Yeast CCT catalyses the folding of yeast ACT1p and human beta-actin with nearly identical rate constants and yields. The results from this controlled CCT-actin folding assay are consistent with a model where CCT and Ac(I) are in a binding pre-equilibrium with a rate-limiting binding step, followed by a faster ATP-driven processing to native actin. In this pure in vitro system, the human beta-actin mutants, D244S and G150P, show impaired folding behaviour in the manner predicted by our sequence-specific recognition model for CCT-actin interaction.

Links

PubMed Online version:10.1016/j.jmb.2006.05.003

Keywords

Actins/chemistry; Actins/metabolism; Adenosine Triphosphate/analogs & derivatives; Adenosine Triphosphate/pharmacology; Chaperonin Containing TCP-1; Chaperonins/chemistry; Chaperonins/isolation & purification; Chaperonins/metabolism; Escherichia coli/genetics; Humans; Mutation/genetics; Protein Binding; Protein Biosynthesis/genetics; Protein Conformation/drug effects; Protein Folding; Saccharomyces cerevisiae/metabolism; Saccharomyces cerevisiae Proteins/chemistry; Saccharomyces cerevisiae Proteins/isolation & purification; Saccharomyces cerevisiae Proteins/metabolism; Time Factors; Transcription, Genetic

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

YEAST:TCPA

part_of

GO:0005832: chaperonin-containing T-complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

YEAST:TCPA

involved_in

GO:0006457: protein folding

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

YEAST:TCPA

enables

GO:0051082: unfolded protein binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

YEAST:TCPB

part_of

GO:0005832: chaperonin-containing T-complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

YEAST:TCPB

involved_in

GO:0006457: protein folding

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

YEAST:TCPB

enables

GO:0051082: unfolded protein binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

YEAST:TCPG

part_of

GO:0005832: chaperonin-containing T-complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

YEAST:TCPG

involved_in

GO:0006457: protein folding

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

YEAST:TCPG

enables

GO:0051082: unfolded protein binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

YEAST:TCPD

part_of

GO:0005832: chaperonin-containing T-complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

YEAST:TCPD

involved_in

GO:0006457: protein folding

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

YEAST:TCPD

enables

GO:0051082: unfolded protein binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

YEAST:TCPZ

part_of

GO:0005832: chaperonin-containing T-complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

YEAST:TCPZ

involved_in

GO:0006457: protein folding

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

YEAST:TCPZ

enables

GO:0051082: unfolded protein binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

YEAST:TCPE

part_of

GO:0005832: chaperonin-containing T-complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

YEAST:TCPE

involved_in

GO:0006457: protein folding

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

YEAST:TCPE

enables

GO:0051082: unfolded protein binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

YEAST:TCPH

part_of

GO:0005832: chaperonin-containing T-complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

YEAST:TCPH

involved_in

GO:0006457: protein folding

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

YEAST:TCPH

enables

GO:0051082: unfolded protein binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

YEAST:TCPQ

part_of

GO:0005832: chaperonin-containing T-complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

YEAST:TCPQ

involved_in

GO:0006457: protein folding

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

YEAST:TCPQ

enables

GO:0051082: unfolded protein binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete


See also

References

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Internal error - GONUTS

GONUTS has been updated to MW1.31 Most things seem to be working but be sure to report problems.

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Internal error

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[9acc33cc842a1c36e04cfd01] /wiki/index.php/PMID:16762366 Error from line 252 of /usr/local/github/wiki-extensions/wiki-extensions-hulab/AnnotationLinks/AnnotationLinks.php: Call to a member function getIdentifier() on null

Backtrace:

#0 /usr/local/github/wiki-extensions/wiki-extensions-hulab/AnnotationLinks/AnnotationLinks.php(70): AnnotationLinks::getQuickGOannotations(string, array)
#1 /var/www/html/go/wiki/includes/parser/Parser.php(3888): AnnotationLinks::renderAnnotations(NULL, array, Parser, PPFrame_DOM)
#2 /var/www/html/go/wiki/includes/parser/Preprocessor_DOM.php(1362): Parser->extensionSubstitution(array, PPFrame_DOM)
#3 /var/www/html/go/wiki/includes/parser/Parser.php(2942): PPFrame_DOM->expand(DOMElement, integer)
#4 /var/www/html/go/wiki/includes/parser/Parser.php(1293): Parser->replaceVariables(string)
#5 /var/www/html/go/wiki/includes/parser/Parser.php(443): Parser->internalParse(string)
#6 /var/www/html/go/wiki/includes/content/WikitextContent.php(323): Parser->parse(string, Title, ParserOptions, boolean, boolean, integer)
#7 /var/www/html/go/wiki/includes/content/AbstractContent.php(516): WikitextContent->fillParserOutput(Title, integer, ParserOptions, boolean, ParserOutput)
#8 /var/www/html/go/wiki/includes/content/ContentHandler.php(1324): AbstractContent->getParserOutput(Title, integer, ParserOptions)
#9 /usr/local/github/wiki-extensions/wiki-extensions-hulab/CirrusSearch/includes/Updater.php(348): ContentHandler->getParserOutputForIndexing(WikiPage, ParserCache)
#10 /usr/local/github/wiki-extensions/wiki-extensions-hulab/CirrusSearch/includes/Updater.php(407): CirrusSearch\Updater::buildDocument(CirrusSearch, WikiPage, CirrusSearch\Connection, integer, integer, integer)
#11 /usr/local/github/wiki-extensions/wiki-extensions-hulab/CirrusSearch/includes/Updater.php(205): CirrusSearch\Updater->buildDocumentsForPages(array, integer)
#12 /usr/local/github/wiki-extensions/wiki-extensions-hulab/CirrusSearch/includes/Updater.php(83): CirrusSearch\Updater->updatePages(array, integer)
#13 /usr/local/github/wiki-extensions/wiki-extensions-hulab/CirrusSearch/includes/Job/LinksUpdate.php(52): CirrusSearch\Updater->updateFromTitle(Title)
#14 /usr/local/github/wiki-extensions/wiki-extensions-hulab/CirrusSearch/includes/Job/Job.php(99): CirrusSearch\Job\LinksUpdate->doJob()
#15 /var/www/html/go/wiki/includes/jobqueue/JobRunner.php(296): CirrusSearch\Job\Job->run()
#16 /var/www/html/go/wiki/includes/jobqueue/JobRunner.php(193): JobRunner->executeJob(CirrusSearch\Job\LinksUpdate, Wikimedia\Rdbms\LBFactorySimple, BufferingStatsdDataFactory, integer)
#17 /var/www/html/go/wiki/includes/MediaWiki.php(1002): JobRunner->run(array)
#18 /var/www/html/go/wiki/includes/MediaWiki.php(988): MediaWiki->triggerSyncJobs(integer, MediaWiki\Logger\LegacyLogger)
#19 /var/www/html/go/wiki/includes/MediaWiki.php(912): MediaWiki->triggerJobs()
#20 /var/www/html/go/wiki/includes/MediaWiki.php(727): MediaWiki->restInPeace(string, boolean)
#21 /var/www/html/go/wiki/includes/MediaWiki.php(750): MediaWiki->{closure}()
#22 /var/www/html/go/wiki/includes/MediaWiki.php(557): MediaWiki->doPostOutputShutdown(string)
#23 /var/www/html/go/wiki/index.php(42): MediaWiki->run()
#24 {main}

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