We acquired remarkably well-resolved mass spectra of the intact lid from
despite the fact that we anticipated that the nine-component endogenous complex would be heterogeneous. From our data we were able to determine the subunit composition of the intact complex, the substoichiometric complexes present in solution, and those generated as a result of our MS/MS approach. Together with chemical cross-linking, our MS approach allowed us to obtain precise additional information regarding the subunit organization of the lid complex. Based on our data we constructed an interaction map of the lid subunits (
). We identified a heterotetrameric core structure formed by Rpn5, Rpn6, Rpn8, and Rpn9, in which Rpn8 is centered. The absence of Rpn11 from this complex shows that it forms after purification and indicates that it is a stable subcomplex in solution. In addition, we revealed that Sem1 binds to both Rpn7 and Rpn3. Considering the small size of Sem1, we assume that Rpn3 and Rpn7 are also interacting. We could also determine that Rpn3 binds Rpn5. Interestingly, we determined a substoichiometric complex of the lid in which Rpn6 is absent. Close inspection of the spectrum suggests that 20%–30% of the purified lid complexes lack Rpn6. The MS/MS results clearly show that both Rpn9 and Rpn12 and, to a lesser extent, Rpn6, readily dissociate from the lid complex. It is established that the extent of interaction between protein subunits as well as their exposure underlies the release of subunits in tandem MS of heteroligomeric complexes [
]. This indicates that these subunits are therefore located at the periphery of the complex. Consistent with this, Rpn9 is known to be a nonessential subunit of the lid [
]. Interestingly, it has been shown previously that Rpn10, which stabilizes the association of the lid and base, interacts with both Rpn9 and Rpn12 [
]. This suggests that Rpn9 and Rpn12 are located near the contact surface between the base and the lid.
An Interaction Map Summarizing the Data Obtained from Both Native MS and Cross-Linking MS Analysis
A very recent study has shown that an
temperature-sensitive mutant grown at restrictive temperatures contained a subcomplex comprising four out of the nine lid components, Rpn5, Rpn8, Rpn9, and Rpn11 [
]. A similar study with an
temperature-sensitive mutant demonstrated the presence of an Rpn5:6:8:9:11 subcomplex [
]. From our cross-linking data, we could not conclude whether Rpn3:7 or Rpn5:9 were anchored to Rpn11. However the results from the
mutants support the scenario in which Rpn5:9 recruits Rpn11 into the Rpn5:6:8:9:11 subcomplex. In addition, the
mutation prevented the incorporation of Rpn3, Rpn7, and Rpn12 into the lid. These results, together with the evidence that Rpn5:6:8:9 exists as a stable independent subcomplex in solution (
), are consistent with the Rpn5:6:8:9 subcomplex forming the scaffolding core of the lid.
Taking together our data with existing data from analysis of mutants [
] and results of previous two-hybrid analysis [
), we propose a detailed interaction map (
). The fact that all the data could be integrated into a single model increases our confidence in the MS analysis. In the model, two clusters become apparent. The first structural cluster includes Rpn5, Rpn6, Rpn8, Rpn9, and Rpn11, in agreement with the cluster suggested previously [
]. Within the second cluster Rpn3, Rpn7, Rpn12, and Sem1 are included, in accord with recent observations [
]. The link between the two subcomplexes is between Rpn5:Rpn3, and not Rpn7 as suggested by the analysis of an
]. These results explain previous observations in
that in cells in which
is deleted the 26S proteasomes misassemble [
]. Moreover, our structural organization indicates that Rpn11 forms extensive interactions with all three subunits, Rpn5, Rpn8, and Rpn9. Furthermore, in our model Rpn6 is opposite Rpn9 and Rpn12. As these may interact with Rpn10 at the lid–base junction, it follows that Rpn6 is exposed to the cellular environment.
Subunit–Subunit Interactions within the 19S Lid from
Structural Organization of the 19S Lid
Given the significant genetic similarity shared between the lid and the COP9 signalosome, we investigated their structural homology.
summarizes the protein–protein interactions identified within the CSN subunits. Out of the 21 interactions detected, nine were observed among the 19S lid complex (
), and eight of the interactions, although not physically recognized, are feasible with our current model, while 4 interactions are not in correspondence with the lid structure. We speculate that the dense web of protein–protein interactions detected within the CSN complex could also be due to the tendency of the two-hybrid system to produce false positives [
]. However, it is interesting to note that six of the nine similar interactions and two of the eight feasible interactions are found within the Rpn5, Rpn6, Rpn8, Rpn9, and Rpn11 cluster. It is also worth noting that a 2-D electron microscopy study [
] did not deduce a common architecture for the two complexes. However, since both particles have similar sizes, show asymmetric arrangements of their subunits, possess a central channel, and share a large number of similar interactions, the two particles are likely to possess some common structural features.
Subunit–Subunit Interactions within the COP9
The major function of the lid is determined by Rpn11, a specialized isopeptidase that tightly couples the deubiquitination and degradation of substrates [
]. So far, no catalytic activity has been described for any other subunit. A recent study suggests that the role of these subunits is of a scaffold for the other binding partners [
], namely Rpn11, and the base subunits as well as the proteolyic substrates and soluble cofactors. Interestingly, Rpn11 is unable to fold as an isolated subunit into a native, active conformation [
]. Neighboring subunit interactions are probably required to position conjugates for cleavage by Rpn11. The multiple protein–protein interactions observed in this study for
Rpn11 are in accordance with this assumption.
Given that the only interaction observed linking the two protein clusters in the lid is between Rpn3 and Rpn5, this implies that there is a flexible hinge region which allows movement necessary for productive interaction of substrates with Rpn11. Taking into consideration the fact that Rpn6 is present at substoichiometric amounts, and that Rpn11 forms extensive protein interactions, it is interesting to speculate that after substrate binding, Rpn6 dissociates to expose active Rpn11. Support for our hypothesis comes from the finding that Rpn6 is essential for assembly of the lid; however,
mutants do not affect the activity of 26S proteasome after assembly [
]. This may imply that Rpn6 acts as a fail-safe mechanism, at least in
ensuring that active sites are exposed only after assembly is completed. Given the high natural abundance of proteasome particles in the cell and the need to prevent indiscriminate proteolysis of proteins, the masking of active sites before full assembly could be an essential regulatory mechanism. We anticipate that further examination of the levels of Rpn6 within the 19S lid complex will validate this scenario.
In summary, the results presented here extend significantly previous models based on pairwise interactions and allow interesting comparison with the structure of the signalosome. Our model is consistent with a core assembly of four subunits that provides a scaffold for recruitment of additional subunits. Also apparent from our analysis is the fact that subunits are assembled into two protein clusters, possibly providing a cleft in which a polyubiquitinated substrate can be accommodated. Based on our finding of substoichiometric quantities of Rnp6, and its position within the complex, we speculate that a regulatory mechanism involving dissociation of Rpn6 is responsible for the exposure of the active site of Rpn11. Overall, therefore, the subunit architecture that we have described will not only benefit further structural analysis but has also provided insight into the function and regulation of the 19S lid.
More generally, the results of this study highlight major advantages over existing approaches for generating interaction maps. For example, it is established that the yeast two-hybrid approach is complicated by the fact that protein interactions are queried pairwise. Interactions of subunits within protein complexes, such as the anaphase-promoting complex, can be underrepresented in two-hybrid datasets because assembly of such complexes may require higher-order interactions between multiple subunits. A second potential problem is that endogenous proteins may facilitate two-hybrid interactions that are not direct, and the presence of endogenous copies of proteins may confuse the analysis [
]. The other existing approach that has been widely applied is to generate mutants for functional analysis of this complex [
]. This method can also be limited by the availability of appropriate mutants, lack of knowledge about how to generate appropriate mutants, and interpreting the effects of the mutations. The fundamental advantage of our approach is that it is carried out with the wild-type endogenous complex, and heterogeneity and subcomplex formation are immediately apparent from the spectra. Moreover, when combined with gas-phase dissociation we are able to probe the composition of protein subcomplexes and reveal peripheral subunits leading to definition of the structural organization of the complex. The fact that the assembly state of this asymmetric complex, with multiple distinct subunits, can be determined and results incorporated into a comprehensive model highlights the tremendous potential of MS. We anticipate that this approach will be used for determining the organization of many other important cellular complexes for which very little structural data exists.