Identification of the Cdc48•20 S Proteasome as an Ancient AAA+ Proteolytic Machine

Abstract

Proteasomes are the major ATP-dependent proteolytic machines in the eukaryotic and archaeal domains of life. To execute protein degradation, the 20S core peptidase combines with the AAA+ ring of the 19S regulatory particle in eukarya or with the AAA+ PAN ring in some archaea. Here, we find that Cdc48 and 20S from the archaeon Thermoplasma acidophilum interact to form a functional proteasome. Cdc48 is an abundant and essential double-ring AAA+ molecular machine ubiquitously present in archaea, where its function has been uncertain, and in eukarya where Cdc48 participates by largely unknown mechanisms in diverse cellular processes including multiple proteolytic pathways. Thus, proteolysis in collaboration with the 20S peptidase may represent an ancestral function of the Cdc48 family.

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

“…For mmCdc48 YY/ΔN -ta20S, V max and K M for GFP-ssrA degradation (Fig. 4C) were similar to values for taCdc48 ΔN -ta20S degradation of this substrate (4). Thus, the enzymatic machinery required for protein degradation is still present, at least vestigially, in mammalian Cdc48.…”

“…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][2][3][4][5][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.…”

“…1A). Recently, we found that a taCdc48 variant lacking the N domain and C-terminal HbYX tripeptide (taCdc48 ΔN/ΔC3 ) bound ta20S only ∼25-fold less tightly than the parental taCdc48 ΔN enzyme, indicating that non-HbYX residues must also make significant contributions to 20S binding (4). To identify potential non-HbYX contacts, we built a simple model of a Cdc48-20S complex by initially aligning the axis of the hexameric ring of mouse Cdc48/ p97 (Protein Data Bank ID code 3CF1; ∼50% homology with taCdc48; ref.…”

“…1A) (3)(4)(5)(6). In all organisms, the 20S peptidase has a four-ring α 7 β 7 β 7 α 7 structure and uses N-terminal residues of the α subunits to gate entrance of substrates into its degradation chamber (Fig.…”

Architecture and assembly of the archaeal Cdc48⋅20S proteasome

“…Because these movements correlate with bound nucleotide or the presence of human-disease mutations, N-domain movements are thought to be important for regulating Cdc48 function. In archaeal Cdc48, deletion of the N domain increases 20S-binding affinity, increases rates of ATP hydrolysis, and increases unfolding/degradation activity (8,19). Breaking contacts between the N domain and the Significance From microbes to humans, proteolytic machines called proteasomes cleave proteins that are damaged or unnecessary into peptide fragments.…”