Reconfiguration of the proteasome during chaperone-mediated assembly

Abstract

The proteasomal ATPase ring, comprising Rpt1-Rpt6, associates with the heptameric alpha-ring of the proteasome core particle (CP) in the mature proteasome, with the Rpt carboxy-terminal tails inserting into pockets of the alpha-ring(1-4). Rpt ring assembly is mediated by four chaperones, each binding a distinct Rpt subunit(5-10). Here we report that the base subassembly of the Saccharomyces cerevisiae proteasome, which includes the Rpt ring, forms a high-affinity complex with the CP. This complex is subject to active dissociation by the chaperones Hsm3, Nas6 and Rpn14. Chaperone-mediated dissociation was abrogated by a non-hydrolysable ATP analogue, indicating that chaperone action is coupled to nucleotide hydrolysis by the Rpt ring. Unexpectedly, synthetic Rpt tail peptides bound alpha-pockets with poor specificity, except for Rpt6, which uniquely bound the alpha 2/alpha 3-pocket. Although the Rpt6 tail is not visualized within an alpha-pocket in mature proteasomes(2-4), it inserts into the alpha 2/alpha 3-pocket in the base-CP complex and is important for complex formation. Thus, the Rpt-CP interface is reconfigured when the lid complex joins the nascent proteasome to form the mature holoenzyme.