Proteasome inhibitors are widely used as therapeutics and research tools, and typically target one of the three active sites, each present twice in the proteasome complex. An endogeneous proteasome inhibitor, PI31, was identified 30 years ago, but its inhibitory mechanism has remained unclear. Here, we identify the mechanism of Saccharomyces cerevisiae PI31, also known as Fub1. Using cryo-electron microscopy (cryo-EM), we show that the conserved carboxy-terminal domain of Fub1 is present inside the proteasome’s barrel-shaped core particle (CP), where it simultaneously interacts with all six active sites. Targeted mutations of Fub1 disrupt proteasome inhibition at one active site, while leaving the other sites unaffected. Fub1 itself evades degradation through distinct mechanisms at each active site. The gate that allows substrates to access the CP is constitutively closed, and Fub1 is enriched in mutant CPs with an abnormally open gate, suggesting that Fub1 may function to neutralize aberrant proteasomes, thereby ensuring the fidelity of proteasome-mediated protein degradation.

蛋白酶体抑制剂被广泛用作治疗和研究工具，通常靶向三个活性位点之一，每个位点在蛋白酶体复合物中出现两次。30 年前发现了一种内源性蛋白酶体抑制剂 PI31，但其抑制机制仍不清楚。在这里，我们确定了酿酒酵母的作用机制PI31，也称为 Fub1。使用低温电子显微镜 (cryo-EM)，我们发现 Fub1 的保守羧基末端结构域存在于蛋白酶体的桶形核心颗粒 (CP) 内部，它同时与所有六个活性位点相互作用。Fub1 的靶向突变破坏了一个活性位点的蛋白酶体抑制，同时不影响其他位点。Fub1 本身通过每个活动站点的不同机制来避免降解。允许底物进入 CP 的门组成性关闭，并且 Fub1 在门异常打开的突变 CP 中富集，表明 Fub1 可能起到中和异常蛋白酶体的作用，从而确保蛋白酶体介导的蛋白质降解的保真度。