Abstract
The proteasome degrades most superfluous and damaged proteins, and its decline is associated with many diseases. As the proteolytic unit, the 20S proteasome is assembled from 28 subunits assisted by chaperones PAC1/2/3/4 and POMP; then, it undergoes the maturation process, in which the proteolytic sites are activated and the assembly chaperones are cleared. However, mechanisms governing the maturation remain elusive. Here, we captured endogenous maturation intermediates of human 20S proteasome, which are low abundance and highly dynamic, and determined their structures by cryo-electron microscopy. Through structure-based functional studies, we identified the key switches that remodel and activate the proteolytic sites. Our results also revealed that the POMP degradation is tightly controlled by a dual-checking mechanism, while the α5 subunit senses POMP degradation to induce PAC1/2 release, achieving the full maturation. These findings elucidate mechanisms directing and safeguarding the proteasome maturation and set basis for building proteasomes to counteract the decline of protein degradation in aging and disease.