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FIG. 1. Schematic illustration of the role of chaperone rings in ATP-dependent protein folding and unfolding/degradation in prokaryotic and eukaryotic cells. Protein folding chaperonins are illustrated in the upper portion of each “cell,” and proteolytic chaperones and the associated proteolytic cylinders are shown in the lower portion. In the case of the prokaryotic Clp components, the homohexameric ATPases, ClpA or ClpX, form coaxial associations with the termini of the double ring cylindrical serine protease, ClpP, delivering recognized substrates to it for degradation (see text). In the absence of association with ClpP, however, ClpA or ClpX can mediate disassembly of oligomeric substrate proteins, exemplified by ClpX-mediated disassembly of the MuA transposase tetramer. Note the two chaperonin classes in the eukaryotic cell (cytosolic and mitochondrial). In the case of the eukaryotic proteasome, the general pathways of ubiquitination to direct proteins for degradation by the proteasome are shown. Not shown is the presence of the proteasome in the nuclear compartment, where similar pathways of turnover appear to be operative.

oligomeric dissociation, may be governed by whether the ring is associated with a cognate proteolytic cylinder.

Notably, other ring-shaped proteolytic assemblies in the cell have covalently linked the ATPase and protease functions in one polypeptide, as in the FtsH bacterial membrane metalloprotease or the related AAA-ATPase containing proteases of the mitochondrial inner membrane, Yta10–12 and Yme1 (refs. 2226; Fig. 1). Joining of the two functions within one polypeptide is not restricted to the membrane proteases; the soluble bacterial protease Lon and its mitochondrial homolog, PIM1, are similarly designed (25). The principles of action of these proteases may be the same as those assemblies composed of distinct chaperone and proteolytic rings, but we confine our discussion here to the latter situation, in which the chaperone moiety is amenable to analysis both on its own and in a binary complex with the proteolytic component.

Architecture-Function Considerations

Both chaperonins and the protease-associating chaperone rings, the latter often referred to as regulatory complexes, are radiajly symmetric (or pseudosymmetric) assemblies of ˜110– 140 Å diameter, housing axial cavities (refs. 6 and 27; Fig. 2). Chaperonins are composed of two back-to-back rings whose axial cavities are blocked at the equatorial “base” of each ring by the collective of COOH termini of the surrounding subunits, which protrude into the central space (28). (The COOH termini are not resolvable crystallographically because of disorder from a GGM repeat sequence, but the collective of termini is visible as a mass in cryoEM.) Thus, chaperonins contain two noncontiguous cavities, 45–65 Å in diameter, one at each end of the cylindrical structure. The cavities are formed by surrounding apical domains, attached on hinges to small intermediate domains, hinged in turn to the equatorial base (Fig. 2). The central cavities have been identified by electron microscopy (EM) and functional studies as the sites of binding of non-native polypeptide, which, at least in the case of the bacterial chaperonin, GroEL, occurs through hydrophobic side chains exposed on the cavity wall (see ref. 29). These side chains apparently bind exposed hydrophobic surfaces specifically present in non-native proteins.

The folding-active state of GroEL is produced when both ATP and the cochaperonin GroES bind to the polypeptide-containing ring; the apical domains of the bound ring undergo large conformational movements, 60° upward rotation and 90° clockwise twisting motion, that move the hydrophobic binding sites away from the cavity, releasing the bound protein into what is now a sequestered space that is “capped” by GroES and enlarged 2-fold in volume (refs. 3 and 30; Fig. 2). The walls of the cavity assume a hydrophilic character that favors burial of hydrophobic residues in the folding substrate protein and exposure of hydrophilic residues, promoting folding to the native state.

Protease-associated chaperone rings also exhibit axial cavities but, in contrast with those of chaperonins, these seem likely to be, in the active state, continuous channels through which recognized substrate proteins can be translocated into the central space of the associated proteolytic cylinder (11). The diameter of such channels is somewhat uncertain, lacking crystallographic resolution so far, but recent cryoEM studies approximate the cavity in bacterial ClpA to 70–80 Å at the widest point, narrowing down to a 10- to 20-Å passageway at the end that interfaces with ClpP (6). For its own part, ClpP, in a stand-alone crystal structure, exhibits a central opening at its terminal ends of ˜10 Å (ref. 31; Fig. 2). This opens into a cavity of >50 Å height and diameter. In the case of the crystal structure of the yeast 20S proteasome (32), there is no detectable axial opening into the chamber, with the NH2 termini of the a-subunits obstructing passage (Fig. 2). This implies a gating action by the ATP-dependent association of the 19S “cap” complex with the proteolytic cylinder. Indeed, in the case of the proteasome, a substitution in the ATP binding site of one of six ATPases in the 19S complex (Rpt2) results in a strong inhibition of the peptidase activity of the proteasome, suggesting that even peptides cannot traverse the channel without involving an ATP-directed gating mechanism (33). The small size and apparent gating of the passageways into the proteolytic cylinders appear likely to exclude the bulk of cellular proteins from the lumen of the proteolytic cyliner. At the same time, a requirement is imposed that proteins must be unfolded before their translocation into the proteolytic cylinder. In fact, ClpA alone has been shown to act as an unfoldase in vitro, globally unfolding a monomeric substrate



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