CATALYTIC COMPONENTS OF PROTEASOMES AND THE REGULATION OF PROTEINASE ACTIVITY

The proteasome (multicatalytic proteinase complex) is a large multimer ic complex which is found in the nucleus and cytoplasm of eukaryotic c ells. It plays a major role in both ubiquitin-dependent and ubiquitin- independent nonlysosomal pathways of protein degradation. Proteasome s ubunits are encoded by members of the same gene family and can be divi ded into two groups based on their similarity to the alpha and beta su bunits of the simpler proteasome isolated from Thermoplasma acidophilu m. Proteasomes have a cylindrical structure composed of four rings of seven subunits. The 26S form of the proteasome, which is responsible f or ubiquitin-dependent proteolysis, contains additional regulatory com plexes. Eukaryotic proteasomes have multiple catalytic activities whic h are catalysed at distinct sites. Since proteasomes are unrelated to other known proteases, there are no clues as to which are the catalyti c components from sequence alignments. It has been assumed from studie s with yeast mutants that beta-type subunits play a catalytic role. Us ing a radiolabelled peptidyl chloromethane inhibitor of rat liver prot easomes we have directly identified RC7 as a catalytic component. Inte restingly, mutants in Pre1, the yeast homologue of RC7, have already b een reported to have defective chymotrypsin-like activity. These resul ts taken together confirm a direct catalytic role for these beta-type subunits. Proteasome activities are sensitive to conformational change s and there are several ways in which proteasome function may be modul ated in vivo. Our recent studies have shown that in animal cells at le ast two proteasome subunits can undergo phosphorylation, the level of which is likely to be important for determining proteasome localizatio n, activity or ability to form larger complexes. In addition, we have isolated two isoforms of the 26S proteinase.