Previous work on the proteasome of S. cerevisiae
focused on the CP, culminating in the solution of its crystal structure (38
). However, CPs fail to degrade physiological substrates of the proteasome, and their activity is not stimulated by ubiquitin or ATP. Thus, substrate selection and other key early steps in protein breakdown by the proteasome must be studied with the holoenzyme form of the complex. Here we report the biochemical characterization of the proteasome holoenzyme from S. cerevisiae
. By amino acid sequence analysis, we directly identified 17 subunits that form the RP of the yeast proteasome. Genes encoding a number of these subunits or their homologs were originally identified through a wide variety of genetic screens (8
), only a few of which were designed to detect proteolysis mutants (14
). These data point to the breadth of the regulatory functions of the proteasome. The assembly of these proteins into the same complex provides a common explanation for the disparate and in many cases unexpected phenotypes. These genetic studies also suggest that substrate-specific effects on protein turnover can result from mutations in any of a large number of RP subunit genes, a suggestion which has interesting mechanistic implications.
Of the known mammalian RP subunits, only S5b/p50.5 (17
) appears absent from yeast. We found no evidence for an S5b homolog in purified yeast proteasomes; in agreement with this result, no clear S5b homologs are identifiable in the yeast genome database. Our survey of yeast proteasome components also did not identify proteins homologous to the proteasome activator PA28 (63
), a result which is similarly supported by the lack of close PA28 homologs in the yeast genome. Yeast proteasomes appear to be more uniform than those of mammals in several ways: they do not appear to associate with PA28-like activator proteins that can replace the RP complex, and each of the 32 known subunits is apparently encoded by a single gene. The heterogeneity of the mammalian proteasome appears to regulate the nature of peptide end products of degradation rather than substrate selection and may be linked to the role of the proteasome in antigen processing (12
). The relevance of subunit interchangeability to antigen processing is best exemplified by the LMP proteins, which interchange with other proteolytically active β subunits of the CP to alter the cleavage site specificity of the proteasome (12
Possible interchangeability among the ATPases is suggested both by their strong sequence similarity to one another and by evidence that the ratio of one ATPase to another in the proteasome may change during the course of programmed cell death in M. sexta
, with possible replacement of one ATPase subunit for another (13
). The simplest model for interchangeability among the ATPases, which has a precedent in prokaryotic ATP-dependent proteases (36
), is that each RP contains a single type of ATPase and thus that the various ATPases define distinct proteasome populations. The results of the His6
tagging experiments shown in Fig. exclude this and related models. The data indicate that the six ATPases of the proteasome are present in the same complex, further suggesting that the subunit composition of the yeast proteasome may be uniform from particle to particle. The presence of six ATPases within a given proteasome is consistent with their assembly into a six-member ATPase ring structure analogous to those found in the simple ATP-dependent proteases of prokaryotes (36
). The same analogy suggests that this ring is situated in contact with the CP and that substrates pass through the center of this ring as they translocate into the CP. This model is consistent with the ATP dependence of proteasome assembly from the RP and CP complexes (3
;unpublished data). A strictly determined site of assembly for each ATPase is suggested both by the coassembly of ATPases into a single particle and by the requirement for each ATPase in yeast (30
The 17 subunit assignments proposed here all have a high degree of confidence. For example, for 29 peptides sequenced, all amino acids assigned were in agreement with predictions from the sequence of the yeast genome. Moreover, most of the subunits identified were homologs of known subunits of the mammalian RP (PA700). However, the existence of additional RP subunits in yeast remains a distinct possibility, which could best be addressed by two-dimensional isoelectric focusing and SDS-PAGE. In particular, the low-molecular-mass region of the one-dimensional gels that we used contained many CP-derived bands, which could comigrate with as-yet-unidentified RP subunits. In mammals, PA700 is a stable complex which has been purified and found to associate with the CP to produce a complex that is competent for the degradation of ubiquitin-protein conjugates (1
). We have also partially purified a particle from yeast that can, when added to CPs, similarly reconstitute the degradation of ubiquitin-protein conjugates (unpublished data). However, it has yet to be established that PA700 is identical to the RP dissociated from purified proteasomes (83
). As suggested above, certain components of the proteasome may be loosely associated and thus underrepresented in purified preparations.
The percentages of identities between sequences of yeast and human homologs of the various RP subunits are given in Table . The ATPases are exceptionally conserved, showing 66 to 76% identity, while identity scores for the non-ATPase Rpn subunits are much lower. The only exception is Rpn11/Mpr1, which is 65% identical between yeast and humans. Interestingly, the amino acid sequence surrounding Cys-117 within Rpn11 shows similarity to sequences flanking the active-site cysteine which serves as the nucleophile in deubiquitinating enzymes (Table ). No other RP subunit thus far identified shows significant similarity to known deubiquitinating enzymes. All known Rpn11 homologs contain extended regions of identity to one another surrounding Cys-117 in Rpn11 (Table ). It is plausible that Rpn11 and its homologs from other species function as a new class of deubiquitinating enzymes (Table , group I), potentially accounting for the deubiquitinating activity detected in preparations of the mammalian PA700 complex (53
). The predicted molecular masses of Rpn11 and its homologs are consistent with estimates based on active-site labeling of the bovine PA700 deubiquitinating factor (54
). However, our proteasome preparations had low activity in several deubiquitination assays (data not shown) (52a
), despite containing apparently intact Rpn11. It is possible that the ubiquitin conjugates tested thus far are not the preferred substrates of Rpn11 and that other substrates will allow the detection of Rpn11-dependent isopeptidase activity in yeast proteasomes.
Lam and coworkers have suggested that isopeptidase activity within the proteasome may serve to inhibit the degradation of certain conjugates by progressively trimming their ubiquitin chains from the distal end (53
). We suggest that proteasomal isopeptidase activity may also, depending on the substrate, accelerate conjugate breakdown by removing ubiquitin groups that prevent translocation of the proteolytic substrate through the channel of the CP. Stimulatory effects of removing ubiquitin groups from the substrate may be particularly dramatic for substrates in which ubiquitin groups are bound to multiple lysine residues within the target protein, rather than being assembled into a single chain. Assuming that the tertiary structure of ubiquitin is too stable to be unfolded by the proteasome, as suggested by structural studies (57
), every ubiquitin group that is directly bound to the substrate is expected to prevent access of the substrate polypeptide to the CP in the region surrounding the ubiquitination site. Deubiquitinating enzymes that reverse such linkages would be expected to facilitate degradation, perhaps accounting for the observed simulatory effects of the deubiquitinating enzyme UCH-3 on in vitro ubiquitin-protein conjugate degradation (42
). Consistent with this hypothesis, such results were obtained with a UbK48R derivative of ubiquitin which is deficient in chain formation.
It is presently unclear whether any of the remaining Rpn subunits possess enzymatic activity, since they lack sequence similarities to known enzymes. They are likely to function in the binding of proteolytic substrates, in the binding of soluble cofactors of the proteasome, or as scaffolding proteins that maintain the architecture of the RP complex. Another possible function is to target the proteasome to specific subcellular sites, although recent photobleaching studies with green fluorescent protein-tagged proteasomes indicated that >90% of proteasomes are freely diffusible in both the nucleus and the cytoplasm (74
). Among Rpn subunits other than Rpn11, the only significant sequence motif identified thus far is a ninefold repeat covering approximately 400 residues in both Rpn1 and Rpn2 (60
). The repeat motif is similar to previously described leucine-rich repeats which have been implicated in specific protein binding. One possible role for these repeats therefore may be binding of the proteolytic substrate, as suggested by Lupas and Baumeister (60
The only Rpn subunit that has been extensively studied is Rpn10/Mcb1 and its homologs in Arabidopsis thaliana
(Mbp1), Drosophila melanogaster
(p54), and humans (S5a). Rpn10 homologs from all of these species are capable of binding multiubiquitin chains in vitro. The universality of this binding interaction strongly suggests its functional significance, and Rpn10/Mcb1/S5a has consequently been proposed to be the multiubiquitin chain receptor of the proteasome (17
). However, yeast mutants in which the RPN10/MCB1
gene has been deleted are viable and competent for the degradation of many ubiquitin conjugates (102
). Only the model substrate ubiquitin–Pro–β-galactosidase has been found to be stabilized in the rpn10
deletion strain. To test whether Rpn10 functions as a ubiquitin receptor, the in vitro ubiquitin chain binding site of Rpn10 was localized. When mutants in which the in vitro ubiquitin chain binding site was deleted were assayed for ubiquitin–Pro–β-galactosidase degradation in vivo, they were found to be fully competent (26
). These data indicate that the role of Rpn10 in protein degradation is probably independent of its ability to bind multiubiquitin chains, at least in S. cerevisiae
. Nonetheless, a comparison of the sequences of Rpn10 and its homologs across eukaryotes indicates that the in vitro ubiquitin chain binding site is stringently conserved evolutionarily (26
), suggesting that it may have a role in proteasome function that has yet to be identified. The mechanistic role of Rpn10/Mcb1 in protein breakdown remains problematic but should emerge from additional genetic analysis.