*2.1. The PAC3-PAC4 Heterodimer Interacts Primarily with* α*5*

To study the biochemical processes involved in proteasome α-subunit assembly mediated by the PAC3-PAC4 heterodimer, we prepared all of the human proteasome α subunits as recombinant proteins. Although protocols to prepare PAC3 and PAC4 as individual recombinant proteins have been reported previously, their heterodimer is rather unstable, unlike the yeast orthologs Pba3 and Pba4 [16,18]. The recombinant PAC4 also has a tendency to form a domain-swapped homodimer [18]. To overcome these problems, we designed and prepared a PAC3-PAC4 heterodimer as a single-chain form, termed scPAC3/4, in which the C-terminus of PAC4 is connected to the N-terminus of PAC3 via a (GGGS)4 liner. All of the recombinant proteins were produced using bacterial expression systems in *Escherichia coli*, and were successfully purified to homogeneity (Figure S1).

To determine which proteasomal α subunits interact with the PAC3-PAC4 heterodimer, we performed in vitro pull-down experiments using these recombinant proteins. In the pull-down assay, His6-tagged scPAC3/4 was applied to Ni2+-charged resin, and subsequently incubated with a mixture of all of the α-subunit proteins. Since α7 spontaneously forms an oligomer that is capable of capturing α6 [19,20], we carried out this experiment both in the absence and in the presence of α7. The pull-down experiments showed that scPAC3/4 reacted with several α subunits including α4, α5, and α6 (Figure 1). Addition of α7 had virtually no impact on the interaction of α6 with scPAC3/4, suggesting that it has a higher affinity for the PAC3-PAC4 heterodimer than for the α7 oligomer. To avoid ambiguity due to overlapping of the Coomassie Brilliant Blue (CBB)-stained bands, we performed the pull-down experiments using α1, α4, α5, and α6 individually. The pull-down assay showed that scPAC3/4 interacted most strongly with α5 and weakly with α4 and α6. By contrast, no interaction was detected between scPAC3/4 and α1. The interaction between α6 and scPAC3/4 appeared to be enhanced in the presence of the other α subunits. Since α5 and α6 occur consecutively in the native α ring, these data suggest that the PAC3-PAC4 heterodimer is important for α5-α6 subcomplex assembly during proteasome α-ring formation.

**Figure 1.** Pull-down experiments between the PAC3-PAC4 heterodimer and proteasome α subunits. The non-tagged α1–α3 and α5–α7 along with 3xFLAG-tagged α4 were mixed with His6-tagged scPAC3/4 immobilized on Ni2<sup>+</sup>-charged Chelating Sepharose beads. The 3xFLAG-tagged α4 was used to avoid the band overlap between α4 and scPAC3/4. After extensive washing, bound proteins were analyzed using CBB staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The 'Input' lane contained all α subunits and His6-scPAC3/4 (0.5 μg each). The SDS-PAGE bands were assigned according to Figure S1a, and the bands originating from the His6-scPAC3/4 and the bound α subunits are labeled.

#### *2.2. The PAC3-PAC4 Heterodimer Acts as Molecular Matchmaker in* α*4-*α*5-*α*6 Assembly*

In order to explore the functional mechanism of the PAC3-PAC4 heterodimer in proteasome assembly involving α4–α6, we investigated the inter-subunit interactions mediated by scPAC3/4. In a pull-down assay, glutathione *S*-transferase (GST)-fused α5 was used as a bait for probing its interactions with the other α subunits, both in the absence and in the presence of scPAC3/4. GST-α5 interacted weakly with α6 in the presence of scPAC3/4, while the other subunits were not reactive with α5 regardless of the presence or absence of scPAC3/4 (Figure 2). In contrast, GST-α5 weakly interacted with α4 regardless of the presence or absence of scPAC3/4 under this assay condition. The results were not influenced by the presence of α7 (Figures 1 and 2).

**Figure 2.** Pull-down experiments between α5 and the other α subunits. The α1–α4, α6, and α7 subunits were mixed with GST-tagged α5 immobilized on Glutathione Sepharose beads. The 3xFLAG-tagged α4 and His6-tagged α6 were used to avoid the overlap of their bands with those of the other α subunits or scPAC3/4. (**a**) Interactions between α5 and the other α subunits in the presence and absence of the PAC3-PAC4 heterodimer. The 'Input1 and 'Input2 lanes contained His6-scPAC3/4 and α1–α6 subunits in the absence and presence of α7, respectively (0.5 μg each). (**b**) Interaction between α5 and the adjacent α subunits, α4 and α6. The 'Input3 lane contained His6-scPAC3/4 and α4–α6 subunits. The pull-down experiment was also performed using an scPAC3/4 mutant with V77S and K80A substitutions in PAC3. Band assignments were carried out according to Figure S1b.

In yeast, the Pba3-Pba4 heterodimer acts as a matchmaker, reinforcing interactions between the α4 and α5 subunits [17]. The results of our pull-down analysis indicated that the PAC3-PAC4 heterodimer interacts with α4, α5, and α6, thereby acting as a molecular matchmaker for these proteasomal subunits. These findings suggest that the functional roles and interactions of this assembly chaperone complex with the proteasomal subunits are evolutionally conserved between yeast and humans.
