Subcomplexes of PA700, the 19 S Regulator of the 26 S Proteasome, Reveal Relative Roles of AAA Subunits in 26 S Proteasome Assembly and Activation and ATPase Activity

Abstract

We have identified, purified, and characterized three subcomplexes of PA700, the 19 S regulatory complex of the 26 S proteasome. These subcomplexes (denoted PS-1, PS-2, and PS-3) collectively account for all subunits present in purified PA700 but contain no overlapping components or significant levels of non-PA700 proteins. Each subcomplex contained two of the six AAA subunits (Rpt1-6) that form the binding interface of PA700 with the 20 S proteasome, the protease component of the 26 S proteasome. Unlike intact PA700, no individual PA700 subcomplex displayed ATPase activity or proteasome activating activity. However, both activities were manifested by ATP-dependent in vitro reconstitution of PA700 from the subcomplexes. We exploited functional reconstitution to define and distinguish roles of different PA700 subunits in PA700 function by selective alteration of subunits within individual subcomplexes prior to reconstitution. Carboxypeptidase treatment of either PS-2 or PS-3, subcomplexes containing specific Rpt subunits previously shown to have important roles in 26 S proteasome assembly and activation, inhibited these processes but did not affect PA700 reconstitution or ATPase activity. Thus, the intact C termini of both subunits are required for 26 S proteasome assembly and activation but not for PA700 reconstitution. Surprisingly, carboxypeptidase treatment of PS-1 also inhibited 26 S proteasome assembly and activation upon reconstitution with untreated PS-2 and PS-3. These results suggest a previously unidentified role for other PA700 subunits in 26 S proteasome assembly and activation. Our results reveal relative structural and functional relationships among the AAA subunits of PA700 and new insights about mechanisms of 26 S proteasome assembly and activation.The 26 S proteasome is a 2,500,000-Da protease complex that degrades polyubiquitylated proteins by an ATP-dependent mechanism (1, 2). The biochemical processes required for this function are divided between two subcomplexes that compose the holoenzyme (3, 4). The first, called 20 S proteasome or core particle, is a 700,000-Da complex that catalyzes peptide bond hydrolysis (5). The second, called PA700 or 19 S regulatory particle, is a 700,000-Da complex that mediates multiple aspects of proteasome function related to initial binding and subsequent delivery of substrates to the catalytic sites of the 20 S proteasome (6). The 20 S proteasome is composed of 28 subunits representing the products of 14 genes arranged in four axially stacked heteroheptameric rings (7,8). Each of the two center ␤ rings contains three different protease subunits that utilize N-terminal threonine residues as catalytic nucleophiles (5,8,9). These residues line an interior lumen formed by the stacked rings and thus are sequestered from interaction with substrates by a shell of 20 S proteasome subunits. PA700 is composed of 20 different subunits. Six of these subunits, termed Rpt1-6, are AAA 2 (ATPases Associated with various cellular Activities) family members that confer ATPase ...

ATP Binding by Proteasomal ATPases Regulates Cellular Assembly and Substrate-induced Functions of the 26 S Proteasome

“…Support for this model includes cellular accumulation of PA700 with Rpt subunits lacking required 20 S binding elements or when 20 S proteasome content is reduced by RNAi (38,39). Moreover, PA700 can be reconstituted in vitro from three subcomplexes in the absence of 20 S proteasome (54). The current data provide additional support for the 20 S-independent model because ATP binding-defective Rpt subunits that are incompetent for 26 S proteasome assembly also accumulated in intact PA700 (Fig.…”

Nucleotide-dependent switch in proteasome assembly mediated by the Nas6 chaperone

“…The intricate nucleotide dependence on the action of the C-domain chaperones is expected to link the mode of chaperone activity to the assembly state of the nascent proteasome, because intermediates in assembly of the Rpt ring are thought to bind, but not hydrolyze, nucleotides (24,37). Therefore, in our model of assembly, the base is formed in the ATPγS state and only transitions to the ATP h state after Rpt ring closure (Fig.…”

“…Contrary to the extensive knowledge of base-CP complex formation (7,12,13,17,(23)(24)(25)(26)(27), mechanistic understanding of base-lid complex formation is lacking. The nine-subunit lid is thought to self-assemble without dedicated chaperone proteins (28)(29)(30)(31), and then joins with the base to form the RP.…”

C Termini of Proteasomal ATPases Play Nonequivalent Roles in Cellular Assembly of Mammalian 26 S Proteasome

“…Significantly, this ATP-dependent in vitro reconstitution requires intact C termini of HbYX residues shown here to mediate cellular 26 S proteasome assembly (24,25). Finally, PA700 can be reconstituted in vitro from purified subassemblies that may represent cellular assembly intermediates (39). Reconstitution of PA700 from subassemblies requires neither intact C termini of Rpt subunits nor 20 S proteasome, and reconstituted PA700, like independently purified PA700, can subsequently bind to 20 S proteasome to form 26 S proteasome.…”