PI31 is a positive regulator of 20S immunoproteasome assembly.

Proteasome inhibitor of 31,000†Da (PI31) is a 20S proteasome-binding protein originally identified as an inhibitor of in vitro 20S proteasome activity. Although recent studies have elucidated a detailed structural basis for this inhibitory activity, the physiological significance of PI31-mediated proteasome inhibition remains uncertain, and multiple alternative cellular roles for PI31 have been described. Here, we report a role for PI31 as a positive regulator for assembly of the 20S immunoproteasome (20Si), a compositionally and functionally distinct isoform of the proteasome that is poorly inhibited by PI31. Genetic ablation of PI31 in mammalian cells had no effect on the cellular content or activity of constitutively expressed proteasomes but reduced the cellular content and activity of interferon-γ-induced immunoproteasomes. This selective effect results from impaired 20Si assembly, evidenced by the accumulation of 20Si assembly intermediates. Our results highlight a distinction in the assembly pathways of constitutive proteasomes and immunoproteasomes and indicate that PI31 plays a chaperone-like role in the selective assembly of 20S immunoproteasomes.