The dynamic nature of the RP–CP interface was first apparent when the substrate translocation channel of the CP was identified and found to assume open and closed states in a regulated manner11,12
. The organization of this extensive interface has remained unknown, despite fragmentary data from many studies based on either the two-hybrid method, crosslinking, or other approaches27,30,33,43–48
. In general, these studies can be only partially reconciled with each other and with our current understanding of the topologies of the Rpt and α rings. In contrast to all previous work, we have used mutagenesis to focus exclusively on the contacts between Rpt tails and the α pockets in intact proteasomes. Thus, we have mapped those contacts that are thought to provide the key connections between the RP and the CP. The final map is complete, self-consistent, and compatible with constraints that derive from the known subunit orders of the two rings.
The RP–CP contact points exhibit several unanticipated features. Most importantly, there is a general asymmetry in the mapping, such that on one side of the Rpt ring we observe fixed contacts, whereas on the other side of the ring the C-terminus of an Rpt exhibits flexible contacts, with the capacity to insert into more than one α pocket. The possibility that the flexibility of crosslink formation is in general a peculiarity of BMOE-induced crosslinking was excluded through using other crosslinking methods, including CuCl2
, in which no linker arm is present (data not shown). As a consequence of the flexibility of Rpt insertion, we did not identify any unoccupied α pocket, although the existence of such a pocket was anticipated based on the Rpt–α subunit symmetry mismatch. Our data suggest a model in which a proteasome sample is composed of distinct sub-populations, each with a different unoccupied pocket. Such subpopulations are likely to interconvert. Thus, an unoccupied pocket, though not fixed or identifiable by crosslinking, may underlie the flexibility in register of Rpt1, Rpt5, and Rpt4. Moreover, the symmetry mismatch between the Rpt and α rings also dictates that the Rpt tail and the α pockets cannot be aligned in such a way that each tail is proximal to a unique pocket (), since the interpocket angle in the α ring is 51° and the tail-tail angle is on average 60°. This problem could be minimized if only two tails are engaged at a given time14
(i.e., those tails associated with ATP-bound subunit), but with the engagement of four tails, some tails would be required to straddle two α pockets.
Interestingly, the asymmetrical character of the RP–CP interface is consistent with existing genetic data in that strong phenotypes tend to cluster towards the fixed half of the ring. Among Rpt tail mutants, the strongest phenotype is that of Rpt6, which is fixed to the α2–α3 pocket, whereas the weakest phenotype belongs to Rpt1, which shows flexibility in its pocket insertion(ref. 29
and S.P., unpublished data). Likewise, among the α subunits the α3 mutants show a far more pronounced phenotype than α7 (ref. 12,13
). All of the fixed tails – Rpt2, Rpt6, and Rpt3 show strong phenotypes, whether in proteasome assembly or gating8,29
The asymmetric character of the RP–CP interface described here may help to explain the misalignment in the axes of the Rpt and α rings observed in electron microscopic analyses of the proteasome. provides one example17,33–35
. Most likely the RP axis fluctuates substantially with respect to the CP49
, the misaligned orientation described by Bohn et al33
being an average of many species. Based on our mapping, the asymmetry arising from this misalignment is well correlated with that of crosslinking; it was concluded that the center of the Rpt ring is displaced in the direction of the α2–α3 pocket33
, which is in the center of the region characterized by fixed crosslinks. Indeed, it is possible that the asymmetry of the CP-RP interface described in this study may also underlie the tilted and misaligned RP-CP orientation in the PAN holoenzyme17
, despite the homomeric nature of PAN and its corresponding α ring.
Although our findings define important parameters, especially regarding the fixed contact points, further aspects of RP–CP interaction require more study. Do Rpt tails that insert into two α pockets have a preferred pocket? Does the choice of pocket influence the functional consequences of tail engagement, such as in the assembly, gating and stability of the proteasome? Does the shift of a tail from one pocket to another occur primarily as a consequence of ATP hydrolysis? Is it true that any one of four distinct pockets can be unoccupied in a given proteasome (α4–α5, α5–α6, α6–α7, and α7–α1) or, for example, if α6–α7 is not occupied by Rpt4 is it always occupied by Rpt5?
The crosslinking approach requires the use of α pocket and Rpt tail mutants, which could affect the specificity of insertion. However, the only phenotypes we found were mild. Another caveat to the crosslinking approach is that it monitors the entry of Rpt tails into the α pocket, but not every instance of tail entry is necessarily a functional engagement. An important goal for future work is to determine the functional significance of asymmetry at the interface and whether this a property of the Rpt tails or some other element of the interface. It will be interesting to find mutants in which the symmetry of the interface is altered.
Proposed role of Rpt–CP contacts in Rpt ring assembly
Mapping of the Rpt–CP contacts also provides new information on assembly of the Rpt ring. Previous studies have suggested that Rpt ring assembly in yeast is guided, in part, by pre-existing α rings28,29,32
. Of particular importance to this model is that both α subunit mutants and Rpt mutants show the accumulation of Rpt ring assembly intermediates. The Rpt mutants were single amino acid deletions at the C-termini of Rpt4 and Rpt6, the latter displaying a stronger phenotype. To date, only one CP subunit, α3, has been implicated in Rpt ring assembly32
. This subunit might thus be predicted to directly contact one of the Rpt’s that promote assembly, and indeed the tail of Rpt6 crosslinks specifically to α3. This observation provides further support for the idea that the CP promotes Rpt ring assembly in yeast and provides a foundation for more precise studies of this complex assembly pathway.
The asymmetry of the RP–CP interface may also underlie distinct functional differences among the RP chaperones, which bind near the C-termini of four of the Rpt proteins to assist in assembly of the Rpt ring. Rpn14 and Nas6 have been grouped functionally by genetic criteria, and are found on the fixed side of the Rpt ring. Rpn14 and Nas6 can be distinguished genetically from Hsm3 and Nas2, which lie on the flexible side of the ring24,26,38
. For Rpn14 and Nas6, prior studies suggest that the chaperone–Rpt interaction is competitive with RP–CP binding28,29
. The suggested mechanism involves steric hindrance: insertion of the C-terminal tails of the Rpt proteins may be disfavored by the presence of the chaperones, due to the proximity of the chaperones to the CP when they are bound to Rpt proteins28,29
. A still unidentified release mechanism may obtain for Hsm3 and Nas2 (ref. 25,38,50
and S.P., unpub. data). Thus, insertion of Rpt tails into α pockets of the CP on the flexible side of the ring may be poorly suited to displace the chaperones.
RptC-termini and α subunit N-termini may coevolve
Our data raise the possibility that the C-terminal tails of the Rpt subunits have co-evolved with the N-terminal tails of the α subunits, though they are not in contact with one another. For example, Rpt2 has strong gate-opening ability whereas Rpt4 has not been seen to promote gate opening8,10,29
. This may be attributed to the HbYX motif of Rpt2 and the absence of one in Rpt4 (ref. 8
). But this distinction between Rpt2 and Rpt4 may also result from the nature of the N-terminal tails of the α subunits whose movements they direct. Our crosslinking data indicate that the C-terminal tail of Rpt2, in docking at the α3–α4 pocket, would displace the α3 N-terminal tail from its central position in the closed form of the CP gate (). On the other hand, engagement of the Rpt4 C-terminal tail would potentially lead to displacement of the N-terminal tail of α7. However, deletion of the α7 tail does not open the CP gate, whereas that of α3 does12
. This difference is consistent with the positioning of these tails within the gate, with α3’s tail being the most centrally located of any α subunit and α7’s tail being peripheral (). Rpt4 might also influence the α1 N-terminus, which is similarly peripheral (). More generally, it is striking that the N-termini of the α subunits that show flexible crosslinking all point outwards from the CP in the closed state of the complex, whereas those showing fixed crosslink formation all follow an inward or lateral path (see ).
Figure 8 α-subunit N-terminal tails and the Rpt proteins proposed to direct their movements. Top panel as viewed from the RP–CP interface. The closed form of the CP channel is shown4. The N-terminal tails (residues 10–18 of α1, (more ...)
Coupling of gating to nucleotide hydrolysis
The proteasome undergoes cycles of ATP hydrolysis in both the presence and absence of substrate. Thus, the sensitivity of Rpt-α pocket crosslinking to nucleotide suggests that the tail–pocket interaction is dynamic, a conclusion that appears to apply to each Rpt protein. The low crosslinking efficiencies seen in the presence of ADP do not necessarily indicate complete dissociation of the Rpt tail from α pockets or a complete lack of involvement of ADP-bound Rpt proteins in gating. Indeed, ADP is effective in stabilizing RP-CP association, at least for yeast proteasomes42,51
, although whether this effect is related to the engagement of Rpt tails with the CP is not known. The nucleotide-free form of the ATPase might show the most radical changes in orientation of the C-domain, but we cannot as yet probe that state, because wild-type proteasomes dissociate under such conditions.
In accordance with our finding that Rpt tails engage less strongly with the CP in the presence of ADP rather than ATP or ATPγS, we postulate that, as ATP is hydrolyzed by successive subunits in the Rpt ring, the contacts between the RP and CP undergo a cycle of motions in response. Structural studies of related hexameric ATPases suggest that nucleotide is hydrolyzed in a rotary mechanism52,53
, in which one of the highly populated species contains two ATP molecules and two ADP molecules, with two ATPase subunits not interacting with nucleotide. We favor an analogous model for the proteasome14
. Our finding that ATP and ADP have distinct effects on the strength of interaction between the Rpt C-termini and the α pockets therefore suggests that strong engagement of only a subset of tails, possibility as few as two, is needed for opening of the CP gate. The Rpt tails that strongly activate the gate – Rpt2, Rpt3, and Rpt5 – alternate around the ring. Consequently, the gate would remain open through cycles of ATP hydrolysis that proceed around the ring, even if ADP and unbound Rpt subunits adopt conformations that do not promote engagement of their C-termini.