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

Abstract

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...

The crystal structure of yeast CCT reveals intrinsic asymmetry of eukaryotic cytosolic chaperonins

“…However, because of the absence of antibody labelling data on closed CCT complexes, it is not known formally if actin's so‐called subdomain 2 disengages from Cct4 in the closed state as implied in the early model of the closed state of the CCT–actin complex (Llorca et al , 2001b). We now consider that more complex reconfigurations of the partially folded actin molecule on the apical domain surfaces can occur upon lid‐closure, based upon protease‐protection mapping experiments with yeast CCT–ACT1sub4–PLP2 complexes (McCormack et al , 2009) and spectroscopic analysis of CCT–ACT1–PLP2 folding kinetics (Stuart et al , 2011). The X‐ray structure shown here with its novel interactions between actin and Cct1 and Cct7 supports the model of more complex reconfigurations occurring during the two‐step folding cycle (Stuart et al , 2011).…”

Dual Action of ATP Hydrolysis Couples Lid Closure to Substrate Release into the Group II Chaperonin Chamber

“…A number of studies using archaeal and eukaryotic chaperonins have suggested that ATP binding suffices to close the built-in lid and trigger substrate folding (Iizuka et al, 2003; Llorca et al, 2001; Villebeck et al, 2007; Stuart et al, 2010). Subsequent ATP hydrolysis would serve to reopen the lid and release the folded protein.…”

“…The current model proposes that group II chaperonins do not release the substrate during folding (Gómez-Puertas et al, 2004; Stuart et al, 2010). Instead, ATP binding would cause the apical domains with their bound substrate to move, and this movement mechanically forces substrate folding.…”

Chaperones: needed for both the good times and the bad times

“…Steady progress continues to be made on the structure-function relationships and reaction cycles of chaperones bound to individual client proteins [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. In this issue, however, we return to first principles, and shine the spotlight on the role of chaperones in biomolecular assemblies, as first defined for nucleosomes by Laskey [17].…”