Substrate degradation by the 26S proteasome (478.2)

Abstract

The eukaryotic 26S proteasome utilizes a complex set of coordinated processing steps for the ATP‐dependent degradation of ubiquitin‐tagged substrates. Our cryo‐EM studies reveal important features of the proteasome regulatory particle, including a pronounced spiral‐staircase arrangement of its heterohexameric ATPase ring, that facilitate substrate engagement and degradation initiation. Substrate engagement induces a translocation‐competent conformation, in which the de‐ubiquitinating subunit Rpn11 is repositioned to function as a gatekeeper at the entrance of the processing pore and the ATPase ring adopts a distinct spiral‐staircase configuration, suggesting that highly coordinated ATP‐hydrolysis events drive substrate translocation. Optical tweezers single‐molecule studies of a related protease, ClpXP, further support such a coordinated ATP‐hydrolysis mechanism. Systematic mutational analyses of the proteasome ATPase ring, using a heterologous expression system and in‐vitro reconstitution of 26S holoenzymes, indicate that the six ATPase subunits play distinct roles in substrate engagement and translocation, corresponding to their positions in the spiral‐staircase arrangements of the ATPase ring. Furthermore, structural and mutational studies of Rpn11 provide new mechanistic insights into the translocation‐dependence and regulation of substrate de‐ubiquitination.