The eukaryotic chaperonin containing TCP-1 (CCT) folds the cytoskeletal protein actin. The folding mechanism of this 16 subunit, 1MDa machine is poorly characterised due to the absence of quantitative in vitro assays. We identified phosducin-like protein 2 (PLP2) as an ATP-elutable binding partner of yeast CCT while establishing the CCT interactome. In a novel, in vitro CCT-ACT1 folding assay which is functional under physiological conditions, PLP2 is a stimulatory co-factor. In a single ATP-driven cycle, PLP2-CCT-ACT1 complexes yield 30-fold more native actin than CCT-ACT1 complexes. PLP2 interacts directly with ACT1 through the C-terminus of its thioredoxin fold and the CCT-binding subdomain 4 of actin. The in vitro CCT-ACT1-PLP2 folding cycle of the pre-assembled complex takes 90s at 30(o)C, several times slower than the canonical chaperonin GroEL. The specific interactions between PLP2, CCT and ACT1 in the yeast-component in vitro system and the pronounced stimulatory effect of PLP2 on actin folding is consistent with in vivo genetic approaches demonstrating an essential and positive role for PLP2 in cellular processes involving actin in Saccharomyces cerevisiae. In mammalian systems however several members of the phosducin-like protein family, including human PDCL3, the orthologue of PLP2, have been shown to be inhibitory towards CCT-mediated folding of actin in vivo and in vitro. Here, using a rabbit reticulocyte-derived in vitro translation system we find that inhibition of beta-actin folding by PDCL3 can be relieved by exchanging its acidic C-terminal extension for that of PLP2. It seems that additional levels of regulatory control of CCT activity by this phosducin-like protein have emerged in higher eukaryotes. (258 words).
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|