Order of the proteasomal ATPases and eukaryotic proteasome assembly

Abstract

The 26S proteasome is responsible for a large fraction of the regulated protein degradation in eukaryotic cells. The enzyme complex is composed of a 20S proteolytic core particle (CP) capped on one or both ends with a 19S regulatory particle (RP). The RP recognizes and unfolds substrates and translocates them into the CP. The RP can be further divided into lid and base subcomplexes. The base contains a ring of six AAA+ ATPases (Rpts) that directly abuts the CP and is responsible for unfolding substrates and driving them into the CP for proteolysis. Although 120 arrangements of the six different ATPases within the ring are possible in principle, they array themselves in one specific order. The high sequence and structural similarity between the Rpt subunits presents special challenges for their ordered association and incorporation into the assembling proteasome. In this review, we discuss recent advances in our understanding of proteasomal RP base biogenesis, with emphasis on potential specificity determinants in ring arrangement, and the implications of the ATPase ring arrangement for proteasome assembly.

Figure 1

Proteasome structure and assembly. (A) The 26S proteasome structure is shown with biochemically defined subcomplexes listed on the left. Assembly chaperones are listed to the right of the subcomplex on which they primarily act. (B) A current model for proteasomal RP assembly is shown, with emphasis on the putative steps in base assembly. The exact placement of Rpn2 and Rpn13 within the base is unknown. They are displayed at the interface between the Nas2 and Rpn14-Nas6 assembly modules based on reported interactions between Rpn2 and Rpt4 [44] and their presence in mammalian complexes containing Rpt3 and Rpt6, but not the other ATPases [33].

Figure 2

Antibodies to FLAG-tagged Rpn2 or Rpn13 efficiently co-precipitate Nas2. Strains expressing the indicated triply FLAG-tagged proteasome subunits from their normal chromosomal loci were immunoprecipitated with anti-FLAG agarose, and copurifying proteins were eluted from the resin with 200 μg/mL 3xFLAG peptide. The eluates were then probed for the presence of the indicated RACs by Western blotting. The presence of Nas2 in the eluates of both Rpn2 and Rpn13 precipitates indicates that both of these subunits coexist in assembly intermediates containing Nas2. Asterisk, a cleavage product of Nas2 formed during immunoprecipitation.

Figure 3

Potential sources of specificity in eukaryotic proteasomal ATPase ring arrangement. (A) The domain structure of the M. jannaschii PAN subunit is shown. Based on high sequence homology of the eukaryotic ATPases to PAN, the Rpts are assumed to contain the same domains. The letter P between the coiled coil and OB fold domains indicates the position of a pivotal proline residue that can assume a cis or trans peptide bond with its preceding amino acid. CTD, C-terminal domain. (B) Potential sources of specificity in eukaryotic ATPase ring arrangement are shown. i, Protein sequences of each ATPase may encode complementary tertiary structures, promoting specific pairings between the Rpts. ii, The RACs may both positively and negatively influence interactions between ATPases. The strategic positioning of each RAC near the interface between Rpt subunits is consistent with a role in regulating heterodimer interactions within the assembling ATPase ring. iii, Other subunits within the base, such as Rpn2, may contain interaction sites with multiple base assembly intermediates and act as a scaffold to bring them together in the proper arrangement. iv, Analogous to (iii), the 20S CP may act as a template or scaffold to position the ATPase subunits appropriately within the forming ring.