The 26S Proteasome

Ubiquitin tags proteins for protein degradation. The ubiquitination requires three different enzymatic activities, a ubiquitin-activating enzyme (El), a ubiquitin-conjugating enzyme (E2 or Ubc) and a ubiquitin ligase (E3). The action of all three enzymes leads to the establishment of a poly-ubiquitin chain on target proteins which are then recognized and proteolyzed by 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. 

NH2) on a substrate protein (iysozyme shown in purpie) by an E3 or ubiquitin iigase, (the muiticoiored SCF compiex). Note that chains of Ub are generated on the substrate, and these are recognized by the 26S proteasome depicted in the upper right at 1/20 scaie. 

Eukaryotic genomes contain information for more than 20 E2s and hundreds of E3s. In contrast to the wealth of components devoted to marking protein substrates for destruction, only one enzyme, the 26S proteasome, has been found to degrade ubiquitylated proteins. However, there is complexity here as well, since the 26S proteasome is an assemblage of at least 30 different subunits. Moreover, there is a growing list of proteins that act as proteasome activators, adapters, or accessory factors. In this chapter I focus on basic biochemical and physiological properties... 

Fig. 9.3. Electron-microscopic reconstructions of the 26S proteasome. Three images of a doubly-capped 26S proteasome are presented to illustrate the positions of the lid and base subcomplexes of the 19S RC and to identify the most probable location of the RC ATPases.

Both the 26S proteasome and the RC hydrolyze all four nucleotide triphosphates, with ATP and CTP preferred over GTP and UTP [58]. Although ATP hydrolysis is required for conjugate degradation, the two processes are not strictly coupled. Complete inhibition of the peptidase activity of the 26S proteasome by calpain inhibitor I has little effect on the ATPase activity of the enzyme. The nucleotidase activities of the RC and the 26S proteasome closely resemble those of E. coli Lon protease, which is composed of identical subunits that possess both proteolytic and nucleotidase activities in the same polypeptide chain. Like the regulatory complex and 26S proteasome, Lon hydrolyzes all four ribonucleotide triphosphates, but not ADP or AMP [18]. 

The 26S proteasome also degrades non-ubiquitylated proteins [71]. The short-lived enzyme ornithine decarboxylase (ODC) and the cell-cycle regulator p21Cip provide well documented examples of ubiquitin-independent proteolysis by the 26S en-... 

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