Maturation of the proteasome core particle induces an affinity switch that controls regulatory particle association

Proteasome assembly is a complex process, requiring 66 subunits distributed over several subcomplexes to associate in a coordinated fashion. Ten proteasome-specific chaperones have been identified that assist in this process. For two of these, the Pba1-Pba2 dimer, it is well established that they only bind immature core particles (CP) in vivo. In contrast, the regulatory particle (RP) utilizes the same binding surface but only interacts with the mature CP in vivo. It is unclear how these binding events are regulated. Here, we show that Pba1-Pba2 binds tightly to immature CP, preventing RP binding. Changes in the CP that occur upon maturation significantly reduce its affinity for Pba1-Pba2, enabling the RP to displace the chaperone. Mathematical modeling indicates that this “affinity switch” mechanism has likely evolved to improve assembly efficiency by preventing the accumulation of stable, non-productive intermediates. Our work thus provides mechanistic insights into a crucial step in proteasome biogenesis.

(a) 2D-gelelectrophoresis of immature CP from a wildtype strain (see Fig. 4C main text) was subjected to immunoblotting. Panels show sequential probing of the membrane for Pba1-Pba2 (top panel), 2 (middle panel) and 7 (lower panel). Spots indicated with an asterisk are signals derived from prior immunoblot, newly obtained signals are consistent with the expected PI and MW of proteins probed for. (b) Mass Spectrometry analysis of spots on 2D gel. Indicated spots were excised and submitted for analysis by MALDI-TOF, spots for which no result was obtained were subsequent analyzed by LC-MS/MS. MALDI-TOF analyses each time identified only one protein with significant score (p<0.05). For LC-MS/MS only the identified proteins that contributed more then >20% to the total sample spectral count are reported to eliminate low abundant background contamination.

Supplementary Figure 4. Model of assembly is robust to variation and changes in parameters.
(a) Assembly yield is robust to variations in RP and CP concentration in the model. These results are from the mathematical model (see main text and other supplemental material). Aside from variations in total RP and CP concentration ([RP] 0 and [CP] 0 , respectively), all the parameters are identical to those used for the "Affinity Switch" model in the main text. The concentration of the chaperone Pba1-Pba2 was held constant at 1 M. Since the CP and RP are at different concentrations in this case, the assembly yield is defined as the concentration of the RP-CP complex divided by the total concentration of RP or CP, depending on which is smaller. We find that near 100% yield is obtained for a wide variety of concentrations of both RP and CP, indicating that the results in Figure 5 of the main text do not depend on a specific set of RP and CP concentrations. (b) Changes in the magnitude of the affinity switch do not qualitatively effect our predictions. Left panel; similar to Figure 5a in the main text, but with an affinity switch that is 100 times instead of 1,000 times. In this model, the K D of Pba1-Pba2 for the immature CP is unchanged at 1 nM, but it binds more tightly to the mature CP (K D = 100 nM). Right panel; as before, now with an affinity switch that is 10,000 times. The K D of the interaction with the immature CP is again unchanged at 1 nM, but the K D of the mature interaction is weaker at 10 M. Note that in both cases, there is a broad range of Pba1-Pba2 concentrations that provides near 100% assembly yields. Modifying the affinity switch by making the K D to the immature form stronger (or weaker) give similar results (data not shown).

Supplementary Note 1. Model for proteasome assembly
The model that we constructed for proteasome assembly draws heavily on the model we previously developed to describe the assembly of ring-like protein complexes 1 . In this case we represented core particle (CP) assembly as the assembly of the  ring, which in eukaryotes is a heteromeric seven-member ring (e.g.  1 - 7 ). The assembly process in this case is derived from the following basic rules: 1. Assembly of the  ring is nucleated by the formation of an  5 : 6 dimer from the respective monomers. In eukaryotic cells, this process is initiated by the binding of the dimeric chaperone Pba3-Pba4 to these two  subunits 2,3 . For simplicity, we kept the role of Pba3-Pba4 implicit, representing its action by allowing the  5 and  6 monomers to spontaneously dimerize.
2. After formation of the  5 : 6 dimer, either the  4 or  7 monomers could bind to the growing ring, generating the  4 : 5 : 6 or  5 : 6 : 7 trimers, respectively. In this model, all  subunits are monomeric until they associate with the "correct" end of the growing ring. All interactions are also considered to be perfectly specific: i.e.  2 can only interact with  1 and  3 (its neighbors in the  ring), and not any other subunits 3 .
3. Completion of the entire  ring is considered an irreversible step. This is due in part to the fact that fully-formed rings are incredibly thermodynamically stable and thus do not tend to dissociate on physiologically relevant time scales 1,4,5 . In CP assembly, completion of the  ring allows the  subunits to begin assembling on the ring; the Pba3-Pba4 chaperone dissociates from the complex after that process begins, and the chaperone Ump1 binds and assists with  ring formation and the eventual maturation of the CP 2,3 . For simplicity we consider CP maturation to occur upon  ring completion.
4. The Pba1-Pba2 chaperone dimer can bind to any complex in the model that includes both  5 and  6 . Binding of this chaperone to any immature CP complex (i.e. any complex that is not a full  ring) occurs with some K D that is considered distinct from the K D of the interaction with the mature CP. This allows us to implement the affinity switch described in the main text.
5. For simplicity, we do not explicitly consider regulatory particle (RP) assembly in this model. All RP molecules are considered fully assembled and functional, since the RP assembly chaperones (e.g. Nas2) prevent binding of CP subunits to immature RP molecules 6 . Interaction of the RP with  monomers is considered to be fairly weak, while interactions with larger  ring complexes is considered to be strong. This latter consideration arises from the fact that the RP can make a large number of contacts with the surface of the  ring 7 , resulting in a fairly stable complex 1,4,5 .
6. Binding of Pba1-Pba2 and the RP to any  complex is considered to be mutually exclusive. In other words, if an  ring subcomplex is bound to Pba1-Pba2, the RP cannot bind; similarly, if the RP is bound to a complex, Pba1-Pba2 cannot bind.
We used a procedure similar to the one we previously described to enumerate the set of chemical species allowed in this model, and the reactions that could occur between them, subject to the rules described above 1 .