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Protein folding: Versatility of the cytosolic TRiC/CCT Michel R. Leroux and F. Ulrich Hartl

Efficient de novo folding of and requires specialized role in folding actins and tubulins in cooperation two molecular chaperones, the chaperonin TRiC (or with GimC, and its more general function in cellular CCT) and its novel cofactor GimC (or prefoldin). Recent folding — and discuss how its unique abilities are likely to studies indicate that TRiC is exquisitely adapted for this be the result of structural adaptations that are lacking in task, yet has the ability to interact with and assist the prokaryotic . folding of numerous other cellular . Evolutionary origins of actins and tubulins Address: Max-Planck-Institut für Biochemie, Department of Cellular Biochemistry, Am Klopferspitz 18A, D-82152 Martinsried, Germany. It is generally thought that the last common ancestor of E-mail: [email protected] organisms in the present three domains of life was a primitive prokaryote on a lineage that split into two Current Biology 2000, 10:R260–R264 branches, giving rise to the bacterial clade and an archaeal 0960-9822/00/$ – see front matter lineage that subsequently divided and established the © 2000 Elsevier Science Ltd. All rights reserved. eukaryotic domain (Figure 1). The rapidly growing number of complete genome sequences have provided A prominent feature of eukaryotic cells that is absent from ample evidence that eukaryotes also harbour genes that prokaryotes — both bacteria and archaea — is an are more closely related to their bacterial rather than extensive cytoskeletal lattice consisting mainly of fil- archaeal counterparts, and that extensive lateral gene aments and -containing microtubules. Actins and transfer between organisms makes it difficult to formulate tubulins are abundant, highly conserved proteins involved a definitive phylogenetic tree [13]. This mixed genetic in processes that are essential and apparently unique to heritage of eukaryotes is commonly believed to have eukaryotes. These include muscle contraction, the resulted from the engulfment of a proteobacterium by a migration of organelles and segregation of chromosomes, proto-eukaryote, followed by extensive transfer of bacter- the stabilization and alteration of cell shape, ameboid ial genes to the host genome [13,14] (Figure 1). View metadata, citationlocomotion, and similar andpapers endocytosis at core.ac.uk and exocytosis. Recent find- brought to you by CORE

ings have shown that the efficient biogenesis of actins and One important point of contention is whether the provided by Elsevier - Publisher Connector tubulins is closely associated with a specialized cellular endosymbiotic event predated or followed the acquisition machinery consisting of two molecular chaperones with no of a cytoskeleton [14]. As only eukaryotic cells are able to direct prokaryotic counterparts. phagocytose large particulate matter, the presence of actin(s) and tubulin(s) — along with some ancillary pro- One of these molecular chaperones, the cylindrical chap- teins — in the eukaryotic ancestor might have facilitated eronin known as TRiC, for ‘TCP-1 ring complex’, or the two endosymbiotic events that produced mitochondria CCT, for ‘cytosolic chaperonin containing TCP-1’, has a and chloroplasts (Figure 1). Furthermore, the mainte- distinct hetero-oligomeric architecture and interacts in a nance and inheritance of an organelle requires the support subunit-specific manner with actins and possibly also of an endoskeleton [14]. These considerations suggest tubulins [1–3]. The second is the recently discovered that a primitive cytoskeleton was in place soon after the eukaryotic GimC, for ‘Genes involved in emergence of the proto-eukaryotic cell from the archaeal microtubule biogenesis complex’, also referred to as lineage, before the primary endosymbiotic event set the prefoldin [4–7]. No bacterial equivalent of GimC has been stage for the radiation of organelle-containing eukaryotes. found, though a protein related to GimC, but with a simplified subunit composition, exists in archaea. In an Tracing back the evolutionary roots of actins and tubulins evolutionary context, the new findings suggest that the is not straightforward. How is it that two of the most dawning of eukaryotes may have been greatly facilitated slowly evolving eukaryotic proteins lack clear counterparts by the co-evolution of these chaperones with proteins in bacteria or archaea, while most metabolic enzymes, for derived from the presumptive ancestral proteins of actin example, have obvious homologues in distantly related and tubulin — FtsA and FtsZ, respectively. organisms [14]? The answer appears to be that homo- logues with weak sequence similarity do exist. The recent There is, however, growing evidence that the substrates of crystal structure of FtsZ shows striking similarity to the eukaryotic chaperonin are not limited to actins and tubulin, despite the limited sequence identity between tubulins, but rather appear to include a considerable frac- the two proteins [15], whereas another cell-division tion of all cytosolic proteins [8–12]. We shall explore these protein, FtsA, bears just enough resemblance to actin to two seemingly contrasting aspects of the chaperonin — its have been uncovered in searches with actin and Hsp70, bb10g02.qxd 04/04/2000 08:29 Page R261

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whose ATP-binding regions are known to have similar Figure 1 three-dimensional structures [14]. Somewhat surprisingly, FtsA and FtsZ are not universally essential for prokaryotic cell division: FtsA appears to be missing from all archaea, FtsZ is absent from at least some archaea, such as Aeropy- rum pernix (a Crenarchaeote), and not all sequenced bacterial genomes include genes for these two proteins (Figure 1). In light of the weak (but significant) structural similarity of FtsA to actin and FtsZ to tubulin, the ances- tral FtsA and FtsZ proteins are likely to have evolved extremely rapidly in the primordial eukaryotic lineage, giving rise to actins and tubulins whose sequences and functions have since been highly conserved in all extant eukaryotes (Figure 1).

Increased eukaryotic chaperonin/GimC complexity It is notable that the pedigrees of two classes of molecular chaperones, the chaperonins and GimC, fit well with the phylogenetic tree of life shown in Figure 1. Chaperonins are double-ringed toroidal protein complexes that assist de novo protein folding in most cellular compartments [16]. A primordial chaperonin gave rise to Group I chaperonins (GroEL) in the bacterial lineage, which are unequivocally the source of the endosymbiotically derived mitochondrial and chloroplast Hsp60 chaperonins (Figure 1). Group I chaperonins are composed of seven identical subunits per ring, and act in cooperation with a ‘capping’ cofactor, Possible evolutionary path from the Last Universal Common Ancestor GroES, which encloses a non-native substrate in the chap- (LUCA) to all extant organisms in the three domains of life — bacteria, eukarya and archaea — and the evolution of chaperonin systems and eronin cavity during the folding cycle [16]. structural proteins. Proteins at the base of the tree represent the ancestors of the chaperonins or structural proteins in question. A different, Group II, chaperonin evolved in the Different colours in protein complexes represent different homologous archaeal–eukaryal lineage. The archaeal chaperonin, or subunits. Arrows indicating gene losses are meant to account for the absence of chaperones and/or structural proteins in various lineages. thermosome, is distantly related to GroEL, and differs in Alternative origins for certain proteins are shown as question marks; for composition and the number of subunits per ring, having example, the common ancestor to GimC may have evolved only in the eight or nine subunits of one or two types [11,12]. The archaeal–eukaryal lineage, or may have been lost in the bacterial eukaryotic cytosolic chaperonin TRiC is closely related to lineage. The transition of ancestral FtsA and FtsZ to actin and tubulin may have been facilitated by co-evolution with the ancestors of the the archaeal chaperonins, but has evolved eight different eukaryotic chaperonin TRiC and GimC. A primitive cytoskeleton would subunit species that form its eight-membered rings [11,12] have allowed more complex cellular dynamics, including the possible (Figure 1). Neither TRiC nor the thermosome use a phagocytotic events that gave rise to mitochondria and chloroplasts GroES-like cofactor; instead, these chaperonins appear to (shown as arrows branching from two different bacterial lineages). See text for further details. have an intrinsic ability to allow or prevent access to their central cavity via extensions lining the opening of the cavity [17,18]. A small-angle neutron scattering analysis The expansion in the number of eukaryotic Group II reported by Gutsche et al. [19] in this issue has revealed, chaperonin subunits presumably occurred rapidly and unexpectedly, that the allosteric regulation of Group II early in the eukaryotic lineage, as all examined eukaryotes chaperonins differs markedly from that of their Group I — including some of the most divergent and presumably counterparts, with the closure of the Thermoplasma aci- ‘ancient’ eukaryotes, such as Trichomonas vaginalis and dophilum thermosome being triggered by ATP hydrolysis, Giardia lamblia (J. Archibald, personal communication) — rather than by ATP binding as with GroEL/GroES. The have the same eight homologous chaperonin genes [12]. phylogeny of GimC appears to mimic that of TRiC. GimC The same is true for the six GimC subunits, with closely is a hexameric capable of assisting related homologues in yeast, Caenorhabditis elegans and protein folding, in association with a chaperonin [4–7]. As mammals [4,5,7]. So the radiation in the number of TRiC with the Group II chaperonins, it is not found in bacteria and GimC subunits may have occurred around the same and has undergone an increase in subunit complexity — time as the rapid evolution of the ancestral FtsA and FtsZ from two to six subunits — in eukaryotes compared with proteins into actins and tubulins (Figure 1). But were it archaea (Figure 1) [4,5,7]. not for the link between the two chaperones and the bb10g02.qxd 04/04/2000 08:29 Page R262

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efficient biogenesis of the cytoskeletal proteins, these dis- subunits. While the biochemical studies are consistent parate observations may have been overlooked. with subdomain 4 of actin interacting with TRiC, no spe- cific interaction with subdomain 2 was observed [2]; the A unique chaperone system for actin/tubulin biogenesis interaction with subdomain 2 may have been missed, but TRiC is essential in the yeast Saccharomyces cerevisiae, as it cannot be ruled out that the δ subunit of TRiC also expected given that two of its substrates are the major interacts with subdomain 1 (see Figure 2 and Figure 3c). cytoskeletal proteins [11,12]. The refolding of actins and Regardless of the details, if the orientation-dependent tubulins from denatured proteins can be achieved, albeit association is indeed critical to the folding of actin, then inefficiently, by incubation with TRiC and ATP; post- we suspect that even the closely related archaeal chaper- chaperonin assembly of α and β tubulins into dimers onin would be unable to accomplish this function. It requires five additional cofactors [11,12]. Similarly, GroEL remains to be established whether tubulin also interacts can bind to, and assist the folding of, a number of non- with specific subunits of TRiC, but it appears likely [2]. native proteins in vitro, including various proteins of eukaryotic origin. This ability has been ascribed to high- The apparently parallel evolution of TRiC and GimC is affinity interactions of GroEL with hydrophobic surfaces probably not coincidental. These two chaperones inter- exposed on a non-native polypeptide, followed by GroES- act physically and cooperate during protein folding in mediated release and encapsulation of the substrate in a vivo; this is reflected in the similar cytoskeletal defects sequestered compartment that is conducive to folding [16]. caused by disrupting chaperonin or GimC function [4–6]. So it is striking that GroEL and mitochondrial chaperonins In yeast, deletion of GimC subunits causes a dramatic can interact with, and release, unfolded β actin and α decrease in the rate of TRiC-mediated actin folding, and tubulin in vitro in an ATP-dependent manner, but cannot ‘leakage’ of non-native forms of actin from the chaper- effect the folding of these proteins [20]. Not surprisingly, onin–GimC system [6]. The precise way in which the expression of actins or tubulins in Escherichia coli cells two chaperones cooperate is unclear, but decidedly dif- results in the production of non-native proteins that accu- ferent from the synergism of GroEL and GroES. Most mulate in inclusion bodies. notably, GimC binds unfolded substrates, whereas GroES plays a more passive role in capping the GroEL The specificity of TRiC in actin and tubulin folding has cylinder [4–7]. In vitro studies of the archaeal GimC recently been addressed. Notably, TRiC appears to bind from Methanobacterium thermoautotrophicum, MtGimC, specific regions within both actins and tubulins, as deter- confirmed its ability to bind to, and stabilize, unfolded mined mainly by binding experiments with fragments of proteins for subsequent folding by a chaperonin [7]. the cytoskeletal proteins. Three distinct sites, encom- Although active at 30°C, MtGimC does not rescue the passing a limited part of the 42 kDa actin polypeptide, microtubule defects of a yeast strain lacking endogenous were found to bind most tightly to TRiC [2] (Figure 2); GimC, suggesting that at least some of the six different these sites contain parts of actin subdomains 1, 3 and 4 subunits of the eukaryotic chaperone may perform (see Figure 3c; the overall orientation of actin is the specialized functions [7]. same as in Figure 2). Interestingly, the interaction of GroEL with the actin fragments appears to be less spe- The available data leave little doubt that the eukaryotic cific [2]. In the case of tubulin, a pronounced affinity of chaperonin TRiC has evolved a hetero-oligomeric archi- TRiC for a highly localized, somewhat surface-exposed tecture with a highly specialized ability to assist the region of this relatively large (50 kDa) protein has been folding of cytoskeletal proteins, and that GimC plays an observed [2,3] (Figure 2). As a cautionary note, these important role in this process. From an evolutionary per- biochemical studies did not address which regions of the spective, it thus seems possible that the transition of a natural folding intermediates of actins and tubulins proto-eukaryotic cell to a eukaryotic cell with a cytoskele- interact with TRiC. ton might have been facilitated by the co-evolution of FtsA and FtsZ with the two chaperones (see also [12]). A landmark paper by Llorca et al. [1] has illuminated the structural basis of the specificity in a TRiC-mediated actin Substrates of the eukaryotic chaperonin–GimC system folding reaction. Cryo-electron microscopic reconstructions Initially, it was believed that TRiC was an actin and of binary complexes between α actin, or a fragment tubulin specific chaperone [11,12,16]. But the list of thereof, and TRiC revealed that the small domain of actin known TRiC substrates now includes firefly luciferase, a — which contains subdomains 1 and 2 — interacts with neurofilament, a viral capsid protein, Gα-transducin, the δ subunit of TRiC, while subdomain 4 of the large cyclin E, myosin II and the von Hippel-Lindau tumour domain contacts either the β or ε subunit of TRiC suppressor protein (VHL) [8–12]. A recent study [8] has (Figure 3). As noted by the authors [1], it is likely that the shown that a wide array of newly synthesized polypep- tips of an open conformation of the ‘U’-shaped actin tides — some 9–15% of all cytosolic proteins — ranging molecule — subdomains 2 and 4 — bind the two TRiC from 30 to 60 kDa in size can be immunoprecipitated bb10g02.qxd 04/04/2000 08:29 Page R263

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Figure 2 Figure 3

Subunit and domain-specific interaction of TRiC with rabbit α actin, as revealed by cryo-electron microscopy. The chaperonin is coloured gold and actin is highlighted in red. (a) Cut-away side view of the chaperonin–α actin complex. (b) Top, slightly tilted view of the chaperonin–α actin complex. (c) TRiC was decorated with TRiC interacts with defined sequences in the cytoskeletal proteins subunit-specific antibodies to determine the geometry-dependent actin and tubulin, as well as the von Hippel-Lindau tumour suppressor interaction of the complete actin molecule or a fragment (subdomain 4, protein (VHL). Three separate TRiC-binding sites in β actin, spanning Sub4) with TRiC (not shown) [1]. The small domain (S) of actin amino acids 125–179 (red), 244–285 (green) and 340–375 (cyan), interacts with the δ subunit of the chaperonin, and subdomain 4 of the were identified by Rommelaere et al. [2]. In the closely related α and β large domain (L) binds either to the β or ε subunit of the tubulins, one distinct region appears to interact specifically with TRiC: chaperonin — that is, positions 1,4 with respect to the δ subunit. it encompasses amino acids 260–321 (cyan) [2,3], as well as another (Figure adapted from [1] with the kind permission of José Valpuesta.) segment identified separately, amino acids 350–380 (red) [3]. The cytosolic chaperonin also interacts with a specific site (amino acids 100–155, cyan) in VHL [9]. uncovered a subunit of GimC, suggesting that GimC coop- erates with TRiC in VHL folding and assembly [9]. Addi- with TRiC in pulse-labeled cells. We can thus expect the tional studies will likely reveal the scope of action of list of known TRiC substrates to grow substantially. No eukaryotic GimC, but judging from the ability of the homologues of the known substrates are detectable in archaeal counterpart to stabilize a variety of unfolded pro- prokaryotes, suggesting that TRiC may be adapted to teins [7], eukaryotic GimC may also bind a wide range of fold certain recalcitrant eukaryotic proteins. Indeed, substrates — possibly the same substrates as TRiC. firefly luciferase cannot be refolded by the GroEL/GroES chaperonin system [16], and it is possible that many Eight different substrate-binding sites in TRiC TRiC substrates would not be productively handled by One major challenge now is to understand how the hetero- Group I chaperonins. oligomeric structure of TRiC can accommodate its special activity in folding substrates such as actins and tubulins, TRiC may also play a role in regulating the assembly of while at the same time retaining a general ability to multimeric proteins. This may be the case with cyclin E, interact with — and presumably assist the folding of — a which has to be folded and bind its partner protein Cdk2 to significant fraction of the eukaryotic cytosolic proteome. be functional, as well as with the hepatitis capsid protein and with VHL. The interaction with VHL is of particular Analysis of a natural set of GroEL substrates with known interest: it was recently pinned down to a 55 residue region structures suggests that Group I chaperonins preferen- of VHL (Figure 2), and shown to be required for the folding tially bind proteins containing two or more domains with of VHL and assembly of the folded protein with its partners αβ folds, which are predicted to expose extensive elongin B and elongin C [9]. Strikingly, a tumorigenic muta- hydrophobic surfaces in their non-native states [20]. tion in VHL prevents its release from the chaperonin and Interestingly, the fraction of cytosolic proteins that incorporation into a functional complex. Interestingly, a interact with GroEL may be comparable to that found to two-hybrid screen for proteins interacting with VHL interact with TRiC [8,21]. The substrate-binding sites on bb10g02.qxd 04/04/2000 08:29 Page R264

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the so-called ‘apical’ domains of GroEL, forming the References 1. Llorca O, McCormack EA, Hynes G, Grantham J, Cordell J, inside ‘rim’ of the cylinder, have been well characterized, Carrascosa JL, Willison KR, Fernandez JJ, Valpuesta JM: Eukaryotic and are known to involve a number of highly conserved, type II chaperonin CCT interacts with actin through specific mostly hydrophobic residues that can generally bind subunits. Nature 1999, 402:693-696. 2. Rommelaere H, De Neve M, Melki R, Vandekerckhove J, Ampe C: hydrophobic regions in unfolded proteins [11,12,16–18]. The cytosolic class II chaperonin CCT recognizes delineated These hydrophobic residues apparently do not match con- hydrophobic sequences in its target proteins. Biochemistry 1999, served hydrophobic residues in Group II chaperonin sub- 38:3246-3257. 3. 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Willison KR: Composition and function of the eukaryotic cytosolic strates, as well as the possibility of fine-tuning its interac- chaperonin-containing TCP-1. In Molecular Chaperones and Folding Catalysts. Edited by Bukau B. Amsterdam: Harwood tions with specific proteins, in a manner not possible with Academic; 1999:555-571. homo-oligomeric chaperonins. 13. Doolittle WF: Phylogenetic classification and the universal tree. Science 1999, 284: 2124-2128. 14. Doolittle RF: The origins and evolution of eukaryotic proteins. Phil From these considerations, one would predict that TRiC Trans Roy Soc Lond [Biol] 1995, 349:235-240. should have a general affinity for non-native proteins, but 15. Löwe J, Amos LA: Crystal structure of the bacterial cell-division protein FtsZ. Nature 1998, 391:203-206. that this affinity should be lower than that of GroEL as a 16. Hartl FU: Molecular chaperones in cellular protein folding. 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Is it comparable to GroES, except that GimC also interacts with the non-native substrate? Advances in this field occur quickly, so stay tuned.

Acknowledgements We thank John M. Archibald, Irina Gutsche and Vishwas R. Agashe for helpful comments on the manuscript.