Assembly of the 26S Proteasome

Three other components that my laboratory has identified and partially purified from Fraction 2 of reticulocytes, termed CF1-CF3, are involved in the degradation of proteins ligated to ubiquitin [24]. These are apparently subcomplexes of the 26S proteasome, a large ATP-dependent protease complex first described by Re-chsteiner and co-workers [25], CF3 is identical to the 20S proteasome core particle [26], while CFl and CF2 may be similar to the base and lid subcomplexes of the 19S regulatory particle of the 26S proteasome, described more recently by the Finley laboratory [27], In hindsight, the reason for finding subcomplexes, rather than the complete 26S complex in Fraction 2 was technical we have routinely prepared Fraction 2 from ATP-depleted reticulocytes [20], under which conditions the 26S proteasome dissociates to its subcomplexes. We found that incubation of the three subcomplexes in the presence of ATP promotes their assembly to the 26S proteasome [24, 26]. The role of ATP in the assembly of the 26S proteasome complex remains unknown. 

Proteasome activity can also be regulated by phosphorylation. For example, Yang et al have shown that phosphorylation events are necessary for the assembly of the 26S proteasome. Several subunits including MSSl, S4, S6, and S12 of the 19S RC have been shown to be phosphorylated. Recently it bas been demonstrated that assembly of the proteasome requires phosphorylation of Rpt6, an ATPase subunit. ... 

Santamaria, P. G., Finley, D., Ballesta, j. P., and Remacha, M. Rpn6p, a proteasome subunit from Saccharomyces cerevisiae, is essential for the assembly and activity of the 26S proteasome. J Biol Chem 2003,... 

Imai, j., Maruya, M., Yashiroda, H., Yahara, 1., and Tanaka, K. The molecular chaperone Hsp90 plays a role in the assembly and maintenance of the 26S proteasome. Embo J 2003, 22, 3557-67. 

The exact cellular process by which the 26S proteasome is assembled remains unknown, but the best evidence suggests that it results from binding of independently assembled 20S proteasome and PA700/19S RP [76]. There is considerable, but incomplete, information about the assembly of the 20S proteasome [77, 78], whereas very little is known about the assembly of PA700. Formation of 26S proteasome from purified 20S proteasome and PA700 can be achieved in vitro by an... 

Structure, assembly and enzymatic mechanism of the 20S complex have been elucidated, but the functional organization of the 26S proteasome is... 

Bedford L, Paine S, Sheppard PW et al. (2010) Assembly, structure, and function of the 26S proteasome. Trends in Cell Biol 20, 391-401. 

Kueucz, E. et al. Assembly of the Drosophila 26S proteasome is accompanied by extensive subunit rearrangements. Biochem J 2002, 365, 527-36. 

Currently, high-resolution EM image reconstructions for the 26S proteasome (Walz et al. 1998), but no atomic-resolution crystallographic data are available for any complex of 20S proteasomes with ATP-dependent activators. The expected assembly of PAN, the archaebacterial AAA(+) activator of proteasomes (Zwickl et al. 1999) into hexamers suggests a symmetry-mismatched complex in archaebacteria. 

Whereas the structure and function of the 20S proteasome have been elucidated in great detail (for review see Baumeister et al 1998), the 19S regulator is understood only dimly at present. Structural studies are hampered by the low intrinsic stability of this assembly and extensive remodelling, which makes it notoriously difficult to obtain homogeneous preparations. Nevertheless, analyses of yeast. Drosophila and human 26S... 

In eukaryotic cells, the 20S proteasome assembles with one or two 19S regulatory complexes (RC) in an ATP-dependent manner to form the 26S proteasome (see Figure 1) (Hough et al. 1987 Ganoth et al. 1988 Driscoll and Goldberg 1990 Peters et al. 1994). The 26S holoenzyme is the most downstream element of the ubiquitin-proteasome pathway. 

Assembly pathways for the RC are virtually unknown. As mentioned above, the ATP-ases interact with one another and complexes containing all six S4 subfamily members have been observed following in vitro synthesis. Impaired synthesis of the yeast lid subunit Rpn6 results in the absence of the entire lid [134], so presumably lid and base subcomplexes assemble independently and associate in the final stages of RC formation cells. In mammalian cells, 26S proteasomes assemble from preformed regulatory complexes and 20S proteasomes [135]. 

S6, and SlOb) and two 20S a-subunits (C8 and C9) are known to be phosphoryl-ated. Phosphorylation appears to be particularly important for 26S proteasome assembly and stability. The kinase inhibitor staurosporine reduces 26S proteasome levels in mouse lymphoma cells [135] and interferon y results in reduced phosphorylation of 20S proteasome a-subunits and decreased 26S proteasome levels [141]. 

ATP-dependent proteases are complex proteolytic machines. They are present in eubacteria, archaebacteria, in eukaryotic organelles and, as the 20S or 26S proteasome, in the eukaryotic cytosol and nucleoplasm. The activators of all known ATP-dependent proteases are related. They all contain an AAA(+) ATPase domain as a module (Neuwald et al. 1999) and are thought to assemble into hexameric particles or, in the case of 26S proteasomes, are present in six variants in the 19S activators (Glickman et al. 1999). Like the ATPases, the proteolytic components of the ATP-dependent proteases form higher order complexes, but unlike for the ATPases, the symmetry of the protease assemblies varies, and the folds of the subunits need not be related. ClpP is a serine protease, FtsH a metalloprotease, and HslV and the proteasomes from archaebacteria and eubacteria are threomne proteases. 

Chen, P. and Hochstrasser, M. 1996. Autocatalytic subunit processing couples active site formation in the 20S proteasome to completion of assembly. Cell 86 961-72. 

---