Quantitative Actin Folding Reactions using Yeast CCT Purified via an Internal Tag in the CCT3/γ Subunit

Pappenberger, Florian; McCormack, Elizabeth A.; Willison, Keith R.

doi:10.1016/j.jmb.2006.05.003

Cited by 62 publications

(95 citation statements)

References 43 publications

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“…While TRiC has been readily purified endogenously from bovine testes (Frydman et al 1992;Leitner et al 2012) and pseudo-exogenously from yeast (Leitner et al 2012;Pappenberger et al 2006), purification of human TRiC has been limited. Expression of all eight subunits exogenously from the cloned genes is difficult.…”

Section: Discussionmentioning

confidence: 99%

“…Purification of endogenous TRiC from rabbit reticulocytes has also been effective (Frydman et al 1994;Gao et al 1992;Nimmesgern and Hartl 1993;Norcum 1996). More recently, purification of exogenously tagged yeast TRiC in yeast has been developed (Dekker et al 2011;Pappenberger et al 2006), along with purification of endogenous yeast TRiC by exogenously tagging an interacting protein (Leitner et al 2012). Co-expression of all eight human CCT subunits in baby hamster kidney cells has been attempted but resulted in very low yields (Machida et al 2012).…”

Section: Introductionmentioning

confidence: 99%

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Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

Knee

Sergeeva

King

2013

Cell Stress and Chaperones

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Archaeal and eukaryotic cytosols contain group II chaperonins, which have a double-barrel structure and fold proteins inside a cavity in an ATP-dependent manner. The most complex of the chaperonins, the eukaryotic TCP-1 ring complex (TRiC), has eight different subunits, chaperone containing TCP-1 (CCT1-8), that are arranged so that there is one of each subunit per ring. Aspects of the structure and function of the bovine and yeast TRiC have been characterized, but studies of human TRiC have been limited. We have isolated and purified endogenous human TRiC from HeLa suspension cells. This purified human TRiC contained all eight CCT subunits organized into double-barrel rings, consistent with what has been found for bovine and yeast TRiC. The purified human TRiC is active as demonstrated by the luciferase refolding assay. As a more stringent test, the ability of human TRiC to suppress the aggregation of human γD-crystallin was examined. In addition to suppressing offpathway aggregation, TRiC was able to assist the refolding of the crystallin molecules, an activity not found with the lens chaperone, α-crystallin. Additionally, we show that human TRiC from HeLa cell lysate is associated with the heat shock protein 70 and heat shock protein 90 chaperones. Purification of human endogenous TRiC from HeLa cells will enable further characterization of this key chaperonin, required for the reproduction of all human cells.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

Knee

Sergeeva

King

2013

Cell Stress and Chaperones

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“…Labeling of Cys 374 with bulky fluorophores has an effect on the local structure of the amino acid within the actin molecule (16), with the label acting as a mutation that destabilizes the actin molecule sufficiently to allow chemical unfolding over a shorter time scale. It should be noted that chemically unfolded actin, not nascent actin, has been used in these assays, and the solution structures of these two denatured actin states, which are unknown, may well differ (12). Nonetheless, binding of the fluorescently labeled actin to DNase I and vitamin D-binding protein and its ability to polymerize demonstrates that neither the introduction of the fluorophore, nor the manner of unfolding of actin, prevents folding of actin to a functional monomer.…”

Section: Conformational Changes At the C Terminus Of Actin Duringmentioning

confidence: 99%

A Two-step Mechanism for the Folding of Actin by the Yeast Cytosolic Chaperonin

Stuart

Leatherbarrow

Willison

2011

Journal of Biological Chemistry

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Actin requires the chaperonin containing TCP1 (CCT), a hexadecameric ATPase essential for cell viability in eukaryotes, to fold to its native state. Following binding of unfolded actin to CCT, the cavity of the chaperone closes and actin is folded and released in an ATP-dependent folding cycle. In yeast, CCT forms a ternary complex with the phosducin-like protein PLP2p to fold actin, and together they can return nascent or chemically denatured actin to its native state in a pure in vitro folding assay. The complexity of the CCTactin system makes the study of the actin folding mechanism technically challenging. We have established a novel spectroscopic assay through selectively labeling the C terminus of yeast actin with acrylodan and observe significant changes in the acrylodan fluorescence emission spectrum as actin is chemically unfolded and then refolded by the chaperonin. The variation in the polarity of the environment surrounding the fluorescent probe during the unfolding/folding processes has allowed us to monitor actin as it folds on CCT. The rate of actin folding at a range of temperatures and ATP concentrations has been determined for both wild type CCT and a mutant CCT, CCT4anc2, defective in folding actin in vivo. Binding of the non-hydrolysable ATP analog adenosine 5-(␤,␥-imino)-triphosphate to the ternary complex leads to 3-fold faster release of actin from CCT following addition of ATP, suggesting a two-step folding process with a conformational change occurring upon closure of the cavity and a subsequent final folding step involving packing of the C terminus to the native-like state.The cytoskeletal protein actin is one of the most highly conserved in all eukaryotes and is involved in many essential cellular processes such as cell motility and cytokinesis. It exists in two forms: at low ionic concentrations, the G-actin monomer is stable, whereas in the presence of KCl, MgCl 2 , or CaCl 2 , and ATP, the F-actin polymer predominates. The actin monomer consists of two domains, the so-called large domain and the small domain, which surround a nucleotide binding cleft and a high affinity divalent cation binding site (1). Actin can be further divided into subdomains, with the N and C termini co-located at the base of subdomain 1 (Fig. 1). G-actin can be unfolded thermally or chemically in the presence of denaturant or EDTA. Studies of the unfolding kinetics of rabbit skeletal muscle ␣-actin (ActA) with EDTA have shown that following loss of the cation and nucleotide from native actin, the actin then unfolds to an intermediate I 3 , which cannot refold spontaneously (2) (Equation 1). Unfolding actin by EDTA treatment allows folding studies to be performed under physiological conditions. In eukaryotic cells, nascent actin is folded by the cytosolic chaperonin containing TCP1 (CCT or TRiC for TCP1-ring complex). CCT is a 1-MDa protein complex made up of two rings, each of which consists of 8 different subunits, known as CCT␣-CCT in mammals and CCT1-CCT8 in yeast. Recent studies of the CCT interactome hav...

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“…There is experimental evidence that strong intermolecular interactions play a role in the course of CCT-actin folding (Hynes & Willison 2000;McCormack et al 2001b;Neirynck et al 2006;Pappenberger et al 2006). Immunoprecipitation of CCT pre-loaded with unfolded actin has identified specific CCT subunits involved in mediating a specific interaction with actin and a complementary b-actin peptide array analysis has mapped the corresponding sites on actin (Hynes & Willison 2000).…”

Section: Strong Actin-cct Interaction Modelmentioning

confidence: 99%

Development of free-energy-based models for chaperonin containing TCP-1 mediated folding of actin

Altschuler

Willison

2008

J. R. Soc. Interface.

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A free-energy-based approach is used to describe the mechanism through which chaperonincontaining TCP-1 (CCT) folds the filament-forming cytoskeletal protein actin, which is one of its primary substrates. The experimental observations on the actin folding and unfolding pathways are collated and then re-examined from this perspective, allowing us to determine the position of the CCT intervention on the actin free-energy folding landscape. The essential role for CCT in actin folding is to provide a free-energy contribution from its ATP cycle, which drives actin to fold from a stable, trapped intermediate I 3 , to a less stable but now productive folding intermediate I 2 . We develop two hypothetical mechanisms for actin folding founded upon concepts established for the bacterial type I chaperonin GroEL and extend them to the much more complex CCT system of eukaryotes. A new model is presented in which CCT facilitates free-energy transfer through direct coupling of the nucleotide hydrolysis cycle to the phases of actin substrate maturation.

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Quantitative Actin Folding Reactions using Yeast CCT Purified via an Internal Tag in the CCT3/γ Subunit

Cited by 62 publications

References 43 publications

Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

A Two-step Mechanism for the Folding of Actin by the Yeast Cytosolic Chaperonin

Development of free-energy-based models for chaperonin containing TCP-1 mediated folding of actin

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