Eukaryotic proteasome assembly is assisted by multiple dedicated chaperones. In yeast, formation of the heteroheptameric ring composed of alpha 1-alpha 7 subunits is promoted by the heterodimeric chaperone Pba3-Pba4. Here we reveal that in the absence of this dimeric chaperone, alpha 2 replaces alpha 4 during alpha-ring assembly, thereby giving rise to a non-productive complex that lacks alpha 4, beta 3, beta 5, beta 6, and beta 7 subunits and aggregates of alpha 4. Furthermore, our structure-guided mutational data demonstrate that the Pba3-Pba4 heterodimer acts as molecular matchmaker reinforcing the interaction between alpha 4 and alpha 5, which is the crucial step in the alpha-ring formation. (C) 2014 Elsevier Inc. All rights reserved.