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teasome somehow aborted before the NH2-terminal transcriptional activation domain enters the cylinder. A resistance of the NH2-terminal domain to unfolding may underlie its resistance to degradation, but there seems also the possibility that removal of a ubiquitin tag from the polypeptide could provide a means of escape, as discussed below. Alternative to partial processing, however, is a model in which initial cleavage by an endopeptidase separates the two domains and is followed by proteasome-mediated degradation of the COOH-terminal fragment (75).

Although the proteolytic system appears generally to be a committed one, it seems to have evolved a fail-safe mechanism or “editor” that prevents inappropriate commitment to turning over potentially active substrate proteins. The PA700 isopeptidase, an integral component of the mammalian 19S cap, enables removal of ubiquitin monomers from polyubiquitinated proteins, affording the chance to rescue those proteins that bear only short lengths of ubiquitin chains (ref. 76; see also ref. 77). The bias against degradation of short chains would predispose the proteasome to preferentially degrade proteins that have efficiently interacted with E3 enzymes to produce processive formation of long chains. Given that polyubiquitin chains are disassembled by the proteasome from their distal ends (76), translocation into the proteolytic cylinder of substrates carrying long polyubiquitin chains is likely to be favored kinetically over chain removal. On the other hand, those proteins with only short ubiquitin chains can be relieved of them and thus protected from proteasomal turnover.

Isopeptidases are also crucial for the recycling of ubiquitin, insofar as ubiquitin itself is not degraded by the proteasome. So, for both aborted and “productive” substrates, there may be an obligatory, but presumably late, step of ubiquitin removal. Indeed, ubiquitin removal is probably also necessary to allow the entire substrate polypeptide to pass through the channel into the proteolytic cylinder because the folded state of ubiquitin is remarkably stable (e.g., ref. 78), and it is thus likely to present a barrier to translocation. In addition, the ubiquitin chain is likely to be inaccessible to translocation because it is anchored to the ubiquitin receptor of the regulatory particle. These considerations suggest that the last key step in the proteasome’s reaction cycle occurs within the regulatory particle and consists in the release of the ubiquitin chain from the substrate. This step is presumably operative forallsubstrates, both “typical” ones as well as “nonsubstrates” that carry too few ubiquitin groups and are released after being edited by the PA700 isopeptidase.

Prospects for Further Mechanistic Understanding

In the short term, we can look forward to crystallographic views of states of the proteolytic chaperone rings, which will allow deductions about ATP-mediated unfolding and translocation, and further mechanistic studies. For both the chaperonin and proteolytic ring systems, however, attention must focus ultimately on the fate of the substrate polypeptide. This represents a challenge in both cases because the substrate occupies, or comes to occupy in the case of the proteolytic machines, a non-native conformation that does not exhibit the structural order and symmetry of the machines themselves. Indeed, substrates seem likely to occupy an ensemble of conformations, as compared with the uniformity from molecule to molecule of the states of the machines. Understanding this system will require facing some of the same problems that the chaperonin system currently confronts related to the location and conformation of substrate during binding and folding, both of which are difficult to examine at high resolution. Spectroscopic approaches seem likely to yield the most definitive answers but will be stretched to their limits to get at these questions.

We thank W.Fenton for critical reading of the manuscript. E.U.W. is supported by a Jane Coffin Childs Fellowship and A.L.H. by the Howard Hughes Medical Institute.

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