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FIG. 2. Architecture of the eukaryotic proteasome and bacterial ClpAP chaperone-protease complexes and of the bacterial GroEL-GroES chaperonin pair. Side views from electron microscopy of the eukaryotic 26S proteasome (Left) and bacterial ClpAP (Center) showing the respective chaperone assemblies associated with the respective proteolytic cylinders (taken from ref. 11). The stoichiometries of the constitutent oligomeric rings are designated by subscripts; note that the eukaryotic proteasome is composed of seven distinct a. subunits and seven distinct ß subunits arranged 2-fold symmetrically to compose the four rings. Shown below are space-filling cutaway images of the proteolytic cylinders, derived from the crystal structures of Wang et al. (31) and Groll et al. (32), with active sites shown as red dots, as well as ribbon diagrams of their entryways, also taken from ref. 11. A space-filling view of the GroEL-GroES-ADP7 asymmetric chaperonin complex is shown (Upper Right), taken from Xu et al. (3), illustrating the differences between GroEL rings in the polypeptide-accepting and folding-active states. The open trans ring of the asymmetric complex exposes hydrophobic residues (shown in yellow) that can capture a non-native polypeptide. Subsequent GroES/ATP binding to the ring with polypeptide replaces this surface with a hydrophilic one (shown in blue), enlarges the cavity 2-fold in volume, and encapsulates the space in which a polypeptide, released from the hydrophobic binding sites, pursues folding in solitary confinement. Below, the rigid body movements of apical (red) and intermediate (green) domains of GroEL that occur on GroES binding are shown, taken from Xu et al. (3). The apical peptide binding surfaces of helices H and I (arrows), as well as an underlying segment, are removed from facing the central cavity to a position rotated upward 60° and twisted 90° clockwise (see text and ref. 3 for details).

protein (79). Translocation through the channel may constitute the first committed step in proteolysis by these ATP-dependent proteases.

In the case of both the bacterial and eukaryotic chaperone components, the rings apposed coaxially to the proteolytic cylinder are composed of six ATPase-containing subunits (6, 33, 34). Considering that the cognate proteolytic cylinders are 7-membered double or quadruple rings (see, e.g., refs. 31, 32, 36), with the exception of six-fold symmetric HslV (35), there is an obvious symmetry mismatch. With such a 6-on-7 interface, the chaperone subunits cannot form a 1-to-1 match with proteolytic subunits in the same way that, for example, GroEL subunits match up exactly with subunits of the GroES cochaperonin partner (3). It is unclear how this unusual and evolutionarily preserved behavior may translate into a functional role. Is it designed to inherently weaken the association between the two components? This seems unlikely, because most chaperone/protease complexes appear to be stable as long as ATP is present. The symmetry mismatch may dispose to rotational sliding or ratcheting of the faces of the respective rings across each other (6). Perhaps it is a manifestation of a mechanism of translocation of substrate protein down the axial channel, such that a polypeptide chain is “spooled” through a narrow opening into the proteolytic chamber by a rotational or ratcheting motion (see, e.g., ref. 36). This model cannot apply toallATP-dependent proteases, however. As mentioned above, the ATPase and proteolytic domains are contained within a single polypeptide in the Lon and membrane-bound metalloproteases, where linking of these domains would prevent relative rotation. Interestingly, in EM images of the eukaryotic proteasome, the two asymmetric 19S complexes are observed in a 2-fold rotational orientation with respect to each other, potentially requiring coupled rotation to satisfy a ratchet model (e.g., ref. 10; see Fig. 2)

In the case of the proteasomal cap structure, not only is there an eight-subunit “base” containing six ATPase subunits, but also an ˜400-kDa “lid” structure, comprising eight subunits in yeast, connected to the base by what looks like a “hinge” in EM images (ref. 37; Fig. 2). When the lid is removed from the yeast proteasome by a mutation eliminating a protein supporting the connection to the base (Rpn10), ubiquitinated proteins can no longer be degraded. Thus, the lid appears to be specifically required for recognition of ubiquitin conjugates. By contrast, with only the base structure remaining attached to the 20S proteasome rings, a nonubiquitinated protein, casein, can still be efficiently degraded (37). These observations would seem to support a model of recognition wherein the lid structure binds the ubiquitin moiety of a ubiquitinated protein while the

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