TY - JOUR T1 - Conserved prolines in the coiled coil-OB domain linkers of proteasomal ATPases facilitate eukaryotic proteasome base assembly JF - bioRxiv DO - 10.1101/2020.11.13.381962 SP - 2020.11.13.381962 AU - Chin Leng Cheng AU - Michael K Wong AU - Yanjie Li AU - Mark Hochstrasser Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/11/14/2020.11.13.381962.abstract N2 - The proteasome is a large protease complex that degrades both misfolded and regulatory proteins. In eukaryotes, the 26S proteasome contains six different AAA+ ATPase subunits, Rpt1-Rpt6, which form a hexameric ring as part of the base subcomplex that drives unfolding and translocation of substrates into the proteasome core. Archaeal proteasomes contain only a single type of ATPase subunit, the proteasome-activating nucleotidase (PAN), which forms a trimer-of-dimers and is homologous to the eukaryotic Rpt subunits. A key PAN proline residue (P91) forms cis and trans peptide bonds in successive subunits around the ring, allowing efficient dimerization through upstream coiled coils. The importance of the equivalent Rpt prolines in eukaryotic proteasome assembly was unknown. We show an equivalent proline is strictly conserved in Rpt3 (in S. cerevisiae, P93) and Rpt5 (P76), well conserved in Rpt2 (P103), and loosely conserved in Rpt1 (P96) in deeply divergent eukaryotes, but in no case is its mutation strongly deleterious to yeast growth. However, the rpt2-P103A, rpt3-P93A, and rpt5-P76A mutations all cause synthetic defects with specific base assembly chaperone deletions. The Rpt5-P76A mutation decreases the levels of the protein and induces a mild proteasome assembly defect. The yeast rpt2-P103A rpt5-P76A double mutant has strong growth defects attributable to defects in proteasome base formation. Several Rpt subunits in this mutant form aggregates that are cleared, at least in part, by the Hsp42-mediated protein quality control (PQC) machinery. We propose that the conserved Rpt linker prolines promote efficient 26S proteasome base assembly by facilitating specific ATPase heterodimerization.CCcoiled coilOBoligonucleotide-bindingPANproteasome-activating nucleotidaseCPcore particleRPregulatory particlecryo-EMcryogenic electron microscopyPQCprotein quality control ER -