THE REGULATORY PARTICLE OF THE SACCHAROMYCES-CEREVISIAE PROTEASOME

The proteasome is a multisubunit protease responsible for degrading pr oteins conjugated to ubiquitin, The 670-kDa core particle of the prote asome contains the proteolytic active sites, which face an interior ch amber within the particle and are thus protected from the cytoplasm. T he entry of substrates into this chamber is thought to be governed by the regulatory particle of the proteasome, which covers the presumed c hannels leading into the interior of the core particle. We have resolv ed native yeast proteasomes into two electrophoretic variants and have shown that these represent core particles capped with one or two regu latory particles. To determine the subunit composition of the regulato ry particle, yeast proteasomes were purified and analyzed by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Resolution of the individual polypeptides revealed 17 distinct proteins, whose id entities were determined by amino acid sequence analysis, Six of the s ubunits have sequence features of ATPases (Rpt1 to Rpt6), Affinity chr omatography was used to purify regulatory particles from various strai ns, each of which expressed one of the ATPases tagged with hexahistidi ne. In all cases, multiple untagged ATPases copurified, indicating tha t the ATPases assembled together into a heteromeric complex. Of the re maining 11 subunits that we have identified (Rpn1 to Rpn3 and Rpn5 to Rpn12), 8 are encoded by previously described genes and 3 are encoded by genes not previously characterized for yeasts. One of the previousl y unidentified subunits exhibits limited sequence similarity with deub iquitinating enzymes. Overall, regulatory particles from yeasts and ma mmals are remarkably similar, suggesting that the specific mechanistic features of the proteasome have been closely conserved over the cours e of evolution.