Alpha-ring Independent Assembly of the 20S Proteasome

Abstract

Archaeal proteasomes share many features with their eukaryotic counterparts and serve as important models for assembly. Proteasomes are also found in certain bacterial lineages yet their assembly mechanism is thought to be fundamentally different. Here we investigate α-ring formation using recombinant proteasomes from the archaeon Methanococcus maripaludis. Through an engineered disulfide cross-linking strategy, we demonstrate that double α-rings are structurally analogous to half-proteasomes and can form independently of single α-rings. More importantly, via targeted mutagenesis, we show that single α-rings are not required for the efficient assembly of 20S proteasomes. Our data support updating the currently held “α-ring first” view of assembly, initially proposed in studies of archaeal proteasomes, and present a way to reconcile the seemingly separate bacterial assembly mechanism with the rest of the proteasome realm. We suggest that a common assembly network underpins the absolutely conserved architecture of proteasomes across all domains of life.

Proteasome Structure and Assembly

“…A lingering question has been why the assembly of archaeal proteasomes seems to depend on initial formation of a full α-ring heptamer, while bacterial proteasome assembly is α ring-independent. This puzzle may have been resolved based on the recent report of a second potential pathway of archaeal proteasome assembly[26]. This pathway is α ring-independent, similar to that of actinobacteria (Figure 2a(i)).…”

“…For the archaeaon Methanococcus maripaludis, the formation of an α ring is not required for efficient assembly of the CP when both M. maripaludis α and β subunits are coexpressed in E. coli . This conclusion is based on an M. maripaludis α-subunit mutant that cannot form a free α ring but is still able to form heterodimers with coexpressed wild-type M. maripaludis β subunits, leading to formation of functional archaeal CPs[26]. The existence of two archaeal CP assembly pathways suggests that proteasome-containing bacterial species, which are relatively rare, may have acquired proteasome genes from ancient archaea but retained only the α ring-independent assembly pathway[26].…”

Putting it all together: intrinsic and extrinsic mechanisms governing proteasome biogenesis

“…Since not all α subunits form rings on their own, H0 are not the only determinants that contribute to α subunit assembly. The available binding energy resulting from the considerable buried surface area between eukaryotic α subunit pairs within a ring (> 2500 Å 2 ) also contributes to α ring formation (Kwon et al 2004b), as do stabilizing salt bridges (Panfair et al 2015). What is not known is how all of these features combined contribute to the order in which α subunits assemble to form an α-ring.…”

“…Two of these "half-proteasomes" can then associate to form a full CP (Kwon et al 2004a;Mayr et al 1998a), which can be subsequently capped by the hexameric ATPase ring. Archaeal α subunits form rings first (Zwickl et al 1994), and these act as a template for β-ring assembly until a half-proteasome is formed, though a bacterial-like assembly pathway is also possible (Panfair et al 2015). In support of such simple, autonomous assembly, heterologous expression of α, β, and ATPase subunits typically results in formation of properly assembled, active proteasomes.…”

“…However, this role is probably not due to Pba1-Pba2 directly preventing two complete α-rings from coming together. Double α-rings interact via a sawtoothed interface mediated primarily by H1 helices (Panfair et al 2015) akin to the interaction between α-and β-rings in the half proteasome (Zwickl et al 1994). However, Pba1-Pba2 binds to the opposite (i.e.…”