Mapping interaction of assembly factor Rpn14 with the proteasome base reveals a bipartite interface and implies ordered remodeling of intersubunit contacts during proteasome biogenesis.

The 26S proteasome is the largest known protease and an essential mediator of targeted protein degradation, a transformative therapeutic modality for human diseases. Assembly of the 26S proteasome from its 66 cognate subunits depends on nine dedicated assembly chaperones. These chaperones generally function by stabilizing fragile assembly intermediates and/or by regulating the order of subunit association. Whereas the basic functional mechanisms of eight of these nine dedicated chaperones have been at least partially elucidated, the function of Rpn14 (PAAF1 in humans) has remained fully enigmatic. Here, we use a combination of genetics, engineered crosslinking coupled with mass spectrometry, and structural modeling to reveal how Rpn14 interacts with the assembling proteasomal ATPase ring. This model refutes previous Rpn14 binding models and identifies several points of inter-protein steric clash that must undergo remodeling during proteasomal regulatory particle subcomplex maturation. We further show that Rpn14 cooperates with nucleotide to stabilize a known assembly intermediate of the proteasomal base subcomplex. Together, our results illuminate the first known function of Rpn14 during proteasome biogenesis, and provide a framework for detailed mechanistic analyses of how specific interfaces within and between proteasomal subcomplexes are remodeled during their assembly.