In-depth Analysis of the Lid Subunits Assembly Mechanism in Mammals.

The 26S proteasome is a key player in the degradation of ubiquitinated proteins, comprising a 20S core particle (CP) and a 19S regulatory particle (RP). The RP is further divided into base and lid subcomplexes, which are assembled independently from each other. We have previously demonstrated the assembly pathway of the CP and the base by observing assembly intermediates resulting from knockdowns of each proteasome subunit and the assembly chaperones. In this study, we examine the assembly pathway of the mammalian lid, which remains to be elucidated. We show that the lid assembly pathway is conserved between humans and yeast. The final step is the incorporation of Rpn12 into the assembly intermediate consisting of two modular complexes, Rpn3-7-15 and Rpn5-6-8-9-11, in both humans and yeast. Furthermore, we dissect the assembly pathways of the two modular complexes by the knockdown of each lid subunit.

PAC1-PAC2 proteasome assembly chaperone retains the core α4-α7 assembly intermediates in the cytoplasm.

The proteasome core particle (CP) is a cytoplasmic and nuclear protease complex and is comprised of two α-rings and two ß-rings stacked in order of αßßα. The assembly of CP proceeds by ordered recruitment of ß-subunits on an α-ring with help of assembly chaperones PAC1-PAC2, PAC3-PAC4, and UMP1. However, the mechanism of α-ring formation remains unsolved. Here, we show that α4, α5, α6, and α7 form a core intermediate as the initial process of α-ring assembly, which requires PAC3-PAC4. α1 and α3 can be incorporated independently into the core α4-α7 intermediate, whereas α2 incorporation is dependent on preceding incorporation of α1. Through these processes, PAC1-PAC2 prevents nonproductive dimerization of α-ring assembly intermediates. We also found that PAC1-PAC2 overrides the effect of nuclear localization signals of α-subunits and retains α-ring assembly intermediates in the cytoplasm. Our results first show a detailed assembly pathway of proteasomal α-ring and explain the mechanism by which CP assembly occurs in the cytoplasm.

Assembly mechanisms of specialized core particles of the proteasome.

The 26S proteasome has a highly complicated structure comprising the 20S core particle (CP) and the 19S regulatory particle (RP). Along with the standard CP in all eukaryotes, vertebrates have two more subtypes of CP called the immunoproteasome and the thymoproteasome. The immunoproteasome has catalytic subunits ß1i, ß2i, and ß5i replacing ß1, ß2, and ß5 and enhances production of major histocompatibility complex I ligands. The thymoproteasome contains thymus-specific subunit ß5t in place of ß5 or ß5i and plays a pivotal role in positive selection of CD8+ T cells. Here we investigate the assembly pathways of the specialized CPs and show that ß1i and ß2i are incorporated ahead of all the other ß-subunits and that both ß5i and ß5t can be incorporated immediately after the assembly of ß3 in the absence of ß4, distinct from the assembly of the standard CP in which ß-subunits are incorporated in the order of ß2, ß3, ß4, ß5, ß6, ß1, and ß7. The propeptide of ß5t is a key factor for this earlier incorporation, whereas the body sequence seems to be important for the earlier incorporation of ß5i. This unique feature of ß5t and ß5i may account for preferential assembly of the immunoproteasome and the thymoproteasome over the standard type even when both the standard and specialized subunits are co-expressed.

The mechanism for molecular assembly of the proteasome.

In eukaryotic cells, the ubiquitin proteasome system plays important roles in diverse cellular processes. The 26S proteasome is a large enzyme complex that degrades ubiquitinated proteins. It consists of 33 different subunits that form two subcomplexes, the 20S core particle and the 19S regulatory particle. Recently, several chaperones dedicated to the accurate assembly of this protease complex have been identified, but the complete mechanism of the 26S proteasome assembly is still unclear. In this review, we summarize what is known about the assembly of proteasome to date and present our group's recent findings on the role of the GET pathway in the assembly of the 26S proteasome, in addition to its role in mediating the insertion of tail-anchored (TA) proteins into the ER membrane.

Involvement of Bag6 and the TRC pathway in proteasome assembly.

The 26S proteasome has an elaborate structure, consisting of 33 different subunits that form the 20S core particle capped by the 19S regulatory particle on either end. Several chaperones that are dedicated to the accurate assembly of this protease complex have been identified, but the mechanisms underlying proteasome biogenesis remain unexplored so far. Here we report that core particle assembly becomes less efficient if the TRC pathway, which mediates insertion of tail-anchored proteins, is defective. We demonstrate that Bag6, a protein in the TRC pathway that is also responsible for the degradation of mislocalized proteins, is not only involved in core particle assembly but also has a key role in efficient regulatory particle assembly by directly associating with precursor regulatory particles. These findings indicate that proteasome assembly is not solely mediated by dedicated chaperones but also depends on general chaperones that preserve protein homeostasis.