Chaperonins are large ring assemblies that assist protein folding to the native state by binding nonnative proteins in their central cavities and then, upon binding ATP, release the substrate protein into a now-encapsulated cavity to fold productively. Two families of such components have been identified: type I in mitochondria, chloroplasts, and the bacterial cytosol, which rely on a detachable "lid" structure for encapsulation, and type II in archaea and the eukaryotic cytosol, which contain a built-in protrusion structure. We discuss here a number of issues under current study. What is the range of substrates acted on by the two classes of chaperonin, in particular by GroEL in the bacterial cytoplasm and CCT in the eukaryotic cytosol, and are all these substrates subject to encapsulation? What are the determinants for substrate binding by the type II chaperonins? And is the encapsulated chaperonin cavity a passive container that prevents aggregation, or could it be playing an active role in polypeptide folding?

The chaperonin GroEL drives its protein-folding cycle by cooperatively binding ATP to one of its two rings, priming that ring to become folding-active upon GroES binding, while simultaneously discharging the previous folding chamber from the opposite ring. The GroEL-ATP structure, determined by cryo-EM and atomic structure fitting, shows that the intermediate domains rotate downward, switching their intersubunit salt bridge contacts from substrate binding to ATP binding domains. These observations, together with the effects of ATP binding to a GroEL-GroES-ADP complex, suggest structural models for the ATP-induced reduction in affinity for polypeptide and for cooperativity. The model for cooperativity, based on switching of intersubunit salt bridge interactions around the GroEL ring, may provide general insight into cooperativity in other ring complexes and molecular machines.

A role in folding of newly translated proteins in the cytosol of eukaryotes has been proposed for t-complex polypeptide-1 (TCP1), although its molecular targets have not yet been identified. Tubulin is a major cytosolic protein whose assembly into microtubules is critical to many cellular processes. Although numerous studies have focused on the expression of tubulin, little is known about the processes whereby newly translated tubulin subunits acquire conformations that enable them to form alpha-beta-heterodimers. We examined the biogenesis of alpha- and beta-tubulin in rabbit reticulocyte lysate, and report here that newly translated tubulin subunits entered a 900K complex in a protease-sensitive conformation. Addition of Mg-ATP, but not nonhydrolysable analogues, released the tubulin subunits as assembly-competent protein with a conformation that was relatively protease-resistant. The 900K complex purified from reticulocyte lysate contained as its major constituent a 58K protein that cross-reacted with a monoclonal antiserum against mouse TCP1. We conclude that TCP1 functions as a cytosolic chaperone in the biogenesis of tubulin.

The cylindrical Hsp100 chaperone ClpA mediates ATP-dependent unfolding of substrate proteins bearing "tag" sequences, such as the 11-residue ssrA sequence appended to proteins translationally stalled at ribosomes. Unfolding is coupled to translocation through a central channel into the associating protease, ClpP. To explore the topology and mechanism of ClpA action, we carried out chemical crosslinking and functional studies. Whereas a tag from RepA protein crosslinked proximally to the flexible N domains, the ssrA sequence in GFP-ssrA crosslinked distally in the channel to a segment of the distal ATPase domain (D2). Single substitutions placed in this D2 loop, and also in two apparently cooperating proximal (D1) loops, abolished binding of ssrA substrates and unfolded proteins lacking tags and blocked unfolding of GFP-RepA. Additionally, a substitution adjoining the D2 loop allowed binding of ssrA proteins but impaired their translocation. This loop, as in homologous nucleic-acid translocases, may bind substrates proximally and, coupled with ATP hydrolysis, translocate them distally, exerting mechanical force that mediates unfolding.

A role in folding newly translated cytoskeletal proteins in the cytosol of eukaryotes has been proposed for t-complex polypeptide 1 (TCP1). In this study, we investigated tubulin and actin biogenesis in Chinese hamster ovary (CHO) cells. When extracts of pulse-labeled cells were analyzed by anion-exchange and size-exclusion chromatography, newly synthesized a-tubulin, (3-tubulin, and actin were observed to enter a large molecular mass complex (=900 kDa). These proteins were released from this complex capable, in the case of tubulin, of forming heterodimers. The large molecular mass complexes coeluted with TCP1 and could be immunoprecipitated by using an anti-TCP1 antibody. These findings demonstrate that there is a cytosolic pathway for folding tubulin and actin in vivo that involves the TCP1 complex.The eukaryotic cytoskeleton composed of intermediate filaments, microtubules, and microfilaments is essential for a variety of cellular processes including mitosis, motility, and maintenance of cell shape (1). Despite its critical importance for cellular function, little is known regarding the biogenesis of its constituent proteins (2-4). For many polypeptides, molecular chaperones and heat shock proteins are essential for proper folding and assembly into oligomeric complexes (5,6). Previous somatic cell genetic studies implicated the 70-kDa heat shock protein (hsp70) and the mitochondrial hsp60 chaperonin (mt-hsp6O) in tubulin folding and microtubule formation, but this model requires tubulin transit into and out of the mitochondria (7).TCP1 complex is an oligomeric particle (=900 kDa) found in the eukaryotic cytosol consisting of t-complex polypeptide 1 (TCP1) and four or five related polypeptides of similar size (55-60 kDa) (8,9) and has recently been proposed to be the cytosolic equivalent to GroEL and mt-hsp60 (10,11). A role for TCP1 complex as a chaperonin in the eukaryotic cytosol is suggested by in vitro studies demonstrating function of the complex in (i) refolding urea-denatured tubulin and actin (12, 13) and (ii) folding newly translated a-and ,B-tubulin in rabbit reticulocyte lysates (14). Our study indicates that in CHO cells newly synthesized a-and 3-tubulin and actin enter a 900-kDa complex that contains TCP1 and that tubulin is released from this complex competent to form heterodimers. Taken together with in vitro studies of tubulin and actin folding (12)(13)(14) = 37 GBq). Radiolabel incorporation was terminated by flooding the plates with complete medium supplemented to 10 mM unlabeled methionine. The labeled cells were extracted in situ on ice (10 min) using 200 ,ul of extraction buffer (FB buffer; 100 mM KCI/10 mM Pipes, pH 6.8/300 mM sucrose/2 mM MgCl2/1 mM EGTA) (4) containing 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 10 ,uM leupeptin. A modified extraction buffer at pH 7.2 with Hepes (15 mM) replacing Pipes (10 mM) was used for immunoprecipitation studies (see below).Immunoblots. CHO cells (=0.8-2 x 108 total cells) were treated with trypsin and extracted in 500 pl o...

SummaryThe chaperonin GroEL assists the folding of nascent or stress-denatured polypeptides by actions of binding and encapsulation. ATP binding initiates a series of conformational changes triggering the association of the cochaperonin GroES, followed by further large movements that eject the substrate polypeptide from hydrophobic binding sites into a GroES-capped, hydrophilic folding chamber. We used cryo-electron microscopy, statistical analysis, and flexible fitting to resolve a set of distinct GroEL-ATP conformations that can be ordered into a trajectory of domain rotation and elevation. The initial conformations are likely to be the ones that capture polypeptide substrate. Then the binding domains extend radially to separate from each other but maintain their binding surfaces facing the cavity, potentially exerting mechanical force upon kinetically trapped, misfolded substrates. The extended conformation also provides a potential docking site for GroES, to trigger the final, 100° domain rotation constituting the “power stroke” that ejects substrate into the folding chamber.

The GroEL/GroES chaperonin system mediates essential kinetic assistance to protein folding by capturing non‐native species in an open ring of GroEL via hydrophobic contacts, preventing aggregation, and then carrying out folding in an encapsulated ring following binding of ATP/GroES. We have been studying the action of cooperative ATP binding within a ring, which mediates initial apical domain movements that both favor substrate protein binding and enable the initial association of GroES. Using an ATP hydrolysis‐defective version of GroEL, short‐time freezing, and cryoEM, the conformational trajectory of the apical domains has been studied. These observations have implications for the movements that follow initial GroES binding, which form the domed folding‐active ring. We have also continued to investigate the nature of folding in the GroES‐encapsulated GroEL ring, assessing active vs. passive behavior, using a number of approaches.

Productive cis folding by the chaperonin GroEL is triggered by the binding of ATP but not ADP, along with cochaperonin GroES, to the same ring as non-native polypeptide, ejecting polypeptide into an encapsulated hydrophilic chamber. We examined the specific contribution of the gamma-phosphate of ATP to this activation process using complexes of ADP and aluminium or beryllium fluoride. These ATP analogues supported productive cis folding of the substrate protein, rhodanese, even when added to already-formed, folding-inactive cis ADP ternary complexes, essentially introducing the gamma-phosphate of ATP in an independent step. Aluminium fluoride was observed to stabilize the association of GroES with GroEL, with a substantial release of free energy (-46 kcal/mol). To understand the basis of such activation and stabilization, a crystal structure of GroEL-GroES-ADP.AlF3 was determined at 2.8 A. A trigonal AlF3 metal complex was observed in the gamma-phosphate position of the nucleotide pocket of the cis ring. Surprisingly, when this structure was compared with that of the previously determined GroEL-GroES-ADP complex, no other differences were observed. We discuss the likely basis of the ability of gamma-phosphate binding to convert preformed GroEL-GroES-ADP-polypeptide complexes into the folding-active state.