Regulation of the proteasome by Hul5 and calmodulin

Abstract

The 26S proteasome participates in the targeted degradation of a myriad of cellular proteins to regulate essential biological processes such as cell division and stress resistance. Unlike most proteases, the proteasome binds, unfolds, and processively degrades proteins into short oligopeptides. Processive degradation by the proteasome is particularly important as partially degraded protein species have the potential to aggregate and cause significant deleterious effects in the cell. The ubiquitin ligase Hul5/UBE3C is the only cellular factor known to promote proteasome processivity, but the mechanisms underlying this activity are unknown. Here, using a combination of cryo-electron microscopy, in vitro biochemistry, mass spectrometry, biolayer interferometry, and yeast genetics, we have uncovered a complex cellular mechanism linking Hul5 and proteasome processivity with calcium signaling and calmodulin (Cmd1), the primary cytosolic calcium sensor of the cell. We have discovered that Hul5 and Cmd1 form a stable complex in vitro and imaged this Hul5/Cmd1 complex bound to the proteasome with cryo-EM. Our cryo-EM data show that Hul5 forms an arch-like structure over the top of the proteasome, where the HECT domain of Hul5 is positioned directly above the substrate entry portal of the proteasome and proximal to the essential deubiquitinating enzyme Rpn11. Remarkably, these Hul5 structures appear to be regulated by calcium and Cmd1, as in the presence of high calcium, the HECT domain (and the majority) of Hul5 is no longer resolved by cryo-EM. We can show that calcium and Cmd1 regulate multiple forms of Hul5 activity at the proteasome in vitro; And notably, we have shown that calcium and Cmd1 regulate degradation of the proteasome processivity substrate Ura3-Pcl5 in vivo. The work presented here provides the first known link between calcium signaling and protein degradation by the proteasome and provides critical insights into the mechanism by which Hul5 promotes proteasome processivity.