Quantitative actin folding reactions using yeast CCT purified via an internal tag in the CCT3/gamma subunit.
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.
Pappenberger
Günter
G
Cancer Research U.K., Centre for Cell and Molecular Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK.
McCormack
Elizabeth A
EA
Willison
Keith R
KR
D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000235:genetics; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013997:Time Factors; D014158:Transcription, Genetic
D000199:Actins; C512172:CCT3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins