Most regulatory and quality control protein degradation in eukaryotes is mediated by the ubiquitin-proteasome system (Finley, 2009
; Ravid and Hochstrasser, 2008
). This system is central to many cellular processes, including cell division, DNA repair, and adaptive immunity, and defects in it contribute to many human diseases. Typically, proteins to be degraded are tagged with a polyubiquitin chain that targets them to the 26S proteasome. The proteasome is a highly conserved 2.6 MDa protease complex consisting of a barrel-shaped 20S core particle (CP) that houses the proteolytic sites, and a 19S regulatory particle (RP) that binds to one or both ends of the CP. The RP confers ATP dependence on proteasomal proteolysis and mediates recognition and recycling of the polyubiquitin tag and unfolding and translocation of substrates into the CP.
The CP comprises four stacked heptameric rings of related subunits. Each outer ring contains seven distinct α subunits while each inner ring is composed of seven different β subunits. No atomic-resolution structure of the RP is available, and the positions of many individual subunits within it are uncertain. Our understanding of its structure to date is derived primarily from protein interaction analyses and medium-resolution electron microscopy (Bohn et al., 2010
; da Fonseca and Morris, 2008
The RP can be separated into two subcomplexes, the lid and base (Glickman et al., 1998
). The base includes a ring of six related AAA+ ATPase (Rpt) subunits (Rpt1–6) that directly abuts the CP and three non-ATPase (Rpn) subunits, Rpn1, Rpn2, and Rpn13. Rpn13 and another RP subunit, Rpn10, are intrinsic polyubiquitin receptors that cooperate with mobile extrinsic ubiquitin receptors to promote ubiquitinated substrate binding to the proteasome.
The lid shares sequence and structural similarities with the COP9 signalosome (CSN) and the eIF3 translation initiation complex. Subunits of these three complexes each contain one of two characteristic motifs: PCI (Proteasome/COP9/Initiation complex) domains or MPN (Mpr1/Pad1, N-terminal) domains (Hofmann and Bucher, 1998
). Both PCI and MPN domains are thought to be protein-protein interaction domains. The proteasome lid contains nine subunits: six PCI domain-containing subunits, Rpn3, Rpn5–7, Rpn9, and Rpn12; two MPN subunits, Rpn8 and Rpn11; and Sem1/Rpn15. Rpn11 bears a variant of the MPN domain, termed MPN+ or JAMM (Maytal-Kivity et al., 2002
), which harbors an essential deubiquitylating activity that cleaves polyubiquitin chains from substrates (Finley, 2009
Although the composition and functions of the proteasome are well documented, how this complex and highly abundant molecular machine is assembled from at least 33 different polypeptides is still poorly understood. Proteasome biogenesis is conserved across species and involves elements of stochastic self-assembly as well as chaperone-mediated subunit assembly. Much progress has been made recently in understanding both CP and RP-base assembly. CP biogenesis proceeds through the assembly of two half-proteasomes, which then dimerize, triggering the autocatalytic cleavage of β-subunit propeptides and yielding active CP (Chen and Hochstrasser, 1996
). CP assembly is facilitated by three proteasome-specific assembly chaperones (Kusmierczyk and Hochstrasser, 2008
). Base assembly is facilitated by four evolutionarily conserved chaperones; chaperone-bound intermediates associate to yield the assembled base precursor (Tomko and Hochstrasser, 2011
In contrast to base assembly, very little information is available on lid biogenesis, although it appears to be able to form independently of the base in both yeast and mammals (Isono et al., 2007
; Kaneko et al., 2009
). The lid can be divided into two modules based on protein-protein interaction data (Sharon et al., 2006
). Module 1 contains subunits Rpn5, Rpn6, Rpn8, Rpn9, and Rpn11, and module 2 consists of Rpn3, Rpn7, Rpn12, and Sem1 (see ). Several lid subcomplexes have been reported in vivo
, including module 1 (Fukunaga et al., 2010
), but the specific steps in lid assembly remain unclear. In yeast, fully formed base and lid complexes are present at low levels; no RP assembly intermediates containing subcomplexes of both the lid and base have been identified. Thus, lid and base probably join only upon complete assembly of each complex. By contrast, a complex consisting of fully formed lid bound to base subunits Rpt3, Rpt6, Rpn2, and the deubiquitylating enzyme Uch37 has been purified from mammals (Thompson et al., 2009
). This suggests that in mammals either lid assembly occurs in conjunction with a subcomplex of base subunits or precedes association with base subunits, as in yeast. In short, neither the pathways and mediator(s) of lid assembly nor the means by which lid-base association is regulated during RP assembly are known.
LP2 and Rpn12 are competent for assembly
Here, we describe a set of yeast lid intermediates, which led to the discovery of several novel features of RP assembly that help explain its high fidelity. Among the identified lid particles (LPs), a species containing only Rpn12 accumulates in all tested lid mutants, suggesting that stable Rpn12 binding to the assembling lid depends on prior incorporation of all other lid subunits. Yeast with rpn12 mutations accumulate a complementary lid intermediate, herein named LP2, containing all lid subunits except Rpn12. Rpn12 and LP2 associate in vitro to form a particle indistinguishable from the proteasomal lid, and neither of these particles can be efficiently incorporated into 26S proteasomes in the absence of the other. The highly conserved Rpn12 C-terminal tail is important both for stable Rpn12 binding to LP2 and for efficient lid-base association. Using site-directed protein crosslinking, we show that the Rpn12 tail contacts subunits in both the lid and base. Further, a C-terminal domain of Rpn12 is sufficient for proteasome formation and cell viability. Our data suggest a hierarchical lid assembly mechanism in which Rpn12 binds LP2 only upon correct assembly of all other lid subunits, and the conserved Rpn12 tail then helps drive lid-base association. Rpn12 incorporation thus acts as an assembly checkpoint linking proper lid assembly and subsequent RP assembly steps.