Bipartite determinants mediate an evolutionarily conserved interaction between Cdc48 and the 20S peptidase.

Proteasomes are essential and ubiquitous ATP-dependent proteases that function in eukarya, archaea, and some bacteria. These destructive but critically important proteolytic machines use a 20S core peptidase and a hexameric ATPase associated with a variety of cellular activities (AAA+) unfolding ring that unfolds and spools substrates into the peptidase chamber. In archaea, 20S can function with the AAA+ Cdc48 or proteasome-activating nucleotidase (PAN) unfoldases. Both interactions are stabilized by C-terminal tripeptides in AAA+ subunits that dock into pockets on the 20S periphery. Here, we provide evidence that archaeal Cdc48 also uses a distinct set of near-axial interactions to bind 20S and propose that similar dual determinants mediate PAN-20S interactions and Rpt(1-6)-20S interactions in the 26S proteasome. Current dogma holds that the Rpt(1-6) unfolding ring of the 19S regulatory particle is the only AAA+ partner of eukaryotic 20S. By contrast, we show that mammalian Cdc48, a key player in cell-cycle regulation, membrane fusion, and endoplasmic-reticulum-associated degradation, activates mammalian 20S and find that a mouse Cdc48 variant supports protein degradation in combination with 20S. Our results suggest that eukaryotic Cdc48 orthologs function directly with 20S to maintain intracellular protein quality control.