Hexameric assembly of the proteasomal ATPases is templated through their C termini

Abstract

Substrates of the proteasome are recognized and unfolded by the regulatory particle (RP), then translocated into the core particle (CP) to be degraded1. A hetero-hexameric ATPase ring, containing subunits Rpt1-Rpt6, is situated within the base subassembly of the RP1. The ATPase ring sits atop the CP, with the Rpt C-termini inserted into pockets in the CP2–6. We have identified a novel function of the Rpt proteins in proteasome biogenesis through deleting the C-terminal residue from each Rpt. Our results indicate that assembly of the hexameric ATPase ring is templated on the CP. We have also identified an apparent intermediate in base assembly, BP1, which contains Rpn1, three Rpts, and Hsm3, a chaperone for base assembly. The Rpt proteins with the strongest assembly phenotypes, Rpt4 and Rpt6, were absent from BP1. We propose that Rpt4 and Rpt6 form a nucleating complex to initiate base assembly, and that this complex is subsequently joined by BP1 to complete the Rpt ring. Our studies show that assembly of the proteasome base is a rapid yet highly orchestrated process.