Structure of the Blm10–20 S Proteasome Complex by Cryo-electron Microscopy. Insights into the Mechanism of Activation of Mature Yeast Proteasomes

Abstract

The 20 S proteasome is regulated at multiple levels including association with endogenous activators. Two activators have been described for the yeast 20 S proteasome: the 19 S regulatory particle and the Blm10 protein. The sequence of Blm10 is 20% identical to the mammalian PA200 protein. Recent studies have shown that the sequences of Blm10 and PA200 each contain multiple HEAT-repeats and that each binds to the ends of mature proteasomes, suggesting a common structural and biochemical function. In order to advance structural studies, we have developed an efficient purification method that produces high yields of stoichiometric Blm10–mature yeast 20 S proteasome complexes and we constructed a three-dimensional (3D) model of the Blm10–20 S complex from cryo-electron microscopy images. This reconstruction shows that Blm10 binds in a defined orientation to both ends of the 20 S particle and contacts all the proteasome α subunits. Blm10 displays the solenoid folding predicted by the presence of multiple HEAT-like repeats and the axial gates on the α rings of the proteasome appear to be open in the complex. We also performed a genetic analysis in an effort to identify the physiological role of Blm10. These experiments, however, did not reveal a robust phenotype upon gene deletion, overexpression, or in a screen for synthetic effects. This leaves the physiological role of Blm10 unresolved, but challenges earlier findings of a role in DNA repair.