Differential Roles of the COOH Termini of AAA Subunits of PA700 (19 S Regulator) in Asymmetric Assembly and Activation of the 26 S Proteasome

Abstract

The 26 S proteasome is an energy-dependent protease that degrades proteins modified with polyubiquitin chains. It is assembled from two multi-protein subcomplexes: a protease (20 S proteasome) and an ATPase regulatory complex (PA700 or 19 S regulatory particle) that contains six different AAA family subunits (Rpt1 to -6). Here we show that binding of PA700 to the 20 S proteasome is mediated by the COOH termini of two (Rpt2 and Rpt5) of the six Rpt subunits that constitute the interaction surface between the subcomplexes. COOH-terminal peptides of either Rpt2 or Rpt5 bind to the 20 S proteasome and activate hydrolysis of short peptide substrates. Simultaneous binding of both COOH-terminal peptides had additive effects on peptide substrate hydrolysis, suggesting that they bind to distinct sites on the proteasome. In contrast, only the Rpt5 peptide activated hydrolysis of protein substrates. Nevertheless, the COOH-terminal peptide of Rpt2 greatly enhanced this effect, suggesting that proteasome activation is a multistate process. Rpt2 and Rpt5 COOH-terminal peptides cross-linked to different but specific subunits of the 20 S proteasome. These results reveal critical roles of COOH termini of Rpt subunits of PA700 in the assembly and activation of eukaryotic 26 S proteasome. Moreover, they support a model in which Rpt subunits bind to dedicated sites on the proteasome and play specific, nonequivalent roles in the asymmetric assembly and activation of the 26 S proteasome.

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

“…This analysis was conducted principally in vitro using isolated peptides corresponding to the C termini of the proteins or with various homomeric protein complexes (20,21,(23)(24)(25). Therefore, to test the relative roles of the three HbYX motifs of the Rpt subunits in cellular assembly of mammalian 26 S proteasome, we repeated the analysis described above with FLAG-Rpt proteins whose HbYX motifs were deleted.…”

“…Support for an important role of HbYX residues in these processes comes from both in vitro and cellular studies. For example, in vitro reconstitution of 26 S proteasome from purified 20 S proteasome and PA700 is abrogated when the HbYX residues of Rpt2 and Rpt5 are enzymatically removed (24). Moreover, Rpt3 lacking its HbYX residues is defective for incorporation into 26 S proteasome in mammalian cells (25).…”

“…We sought to test the hypothesis, based in part on previous in vitro biochemical studies, that intact HbYX residues of Rpt2, Rpt3, and Rpt5, but not corresponding non-HbYX residues of Rpt1, Rpt4, and Rpt6, would be essential for cellular 26 S proteasome assembly and to establish whether the HbYX motifs of different subunits played independent or cooperative roles in the assembly process (24,25). We also sought to evaluate the physiologic significance of our in vitro reconstitution assay of 26 S proteasome from isolated 20 S proteasome and PA700 subcomplexes by determining whether PA700 is formed as an intact complex prior to its cellular assembly into 26 S proteasome.…”

“…Three of the six Rpt subunits (Rpt2, Rpt3, and Rpt5) feature C-terminal residues that conform to an HbYX motif (where Hb is a hydrophobic residue; Y is tyrosine, and X is any amino acid) (20). These residues bind to pockets between specific ␣ subunits of 20 S proteasome (21)(22)(23)(24). Binding of HbYX residues of either of two Rpt subunits (Rpt2 and Rpt5) or of the HbYX residues of PAN, an Rpt subunit ortholog from archaea, induces gate opening, even when these residues are in the form of isolated short peptides corresponding to the C terminus of respective subunits (20,24,25).…”

Asymmetric perturbations of signalling oligomers

“…Its barrel-shaped 20S peptidase is capped by a 19S regulator, a heterohexamer of Rpt subunits. Complex chemical modification and MS studies revealed that binding of 19S is mediated by the C-termini of only two Rpt subunits to dedicated sites on the proteasome and play nonequivalent roles in the asymmetric assembly and activation of the 26S proteasome (Gillette et al, 2008). Cryo-EM has shown that substrate-induced rearrangement of the ATPase subunits results in seven-fold symmetry of the peptidase barrel (Matyskiela et al, 2013).…”