The Regulatory Particle of the Saccharomyces cerevisiae Proteasome

Abstract

The proteasome is a multisubunit protease responsible for degrading proteins conjugated to ubiquitin. The 670-kDa core particle of the proteasome contains the proteolytic active sites, which face an interior chamber within the particle and are thus protected from the cytoplasm. The entry of substrates into this chamber is thought to be governed by the regulatory particle of the proteasome, which covers the presumed channels leading into the interior of the core particle. We have resolved native yeast proteasomes into two electrophoretic variants and have shown that these represent core particles capped with one or two regulatory particles. To determine the subunit composition of the regulatory 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 identities were determined by amino acid sequence analysis. Six of the subunits have sequence features of ATPases (Rpt1 to Rpt6). Affinity chromatography was used to purify regulatory particles from various strains, each of which expressed one of the ATPases tagged with hexahistidine. In all cases, multiple untagged ATPases copurified, indicating that the ATPases assembled together into a heteromeric complex. Of the remaining 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 previously unidentified subunits exhibits limited sequence similarity with deubiquitinating enzymes. Overall, regulatory particles from yeasts and mammals are remarkably similar, suggesting that the specific mechanistic features of the proteasome have been closely conserved over the course of evolution.In eukaryotes, the elimination of many short-lived proteins requires their covalent attachment to ubiquitin (43). This pathway is involved in a wide variety of regulatory mechanisms, with substrates including cyclins and CDK inhibitors (49), membrane proteins such as CFTR (104), p53 (84), NF-B (71), c-Fos, c-Jun, and luminal components of the endoplasmic reticulum (9). Multiubiquitin chains target proteins for degradation by the proteasome, an ϳ2-MDa proteolytic complex (reviewed in references 12, 55, 61, and 80).The mechanism of the proteasome is thought to involve unfolding of a protein substrate and translocation from one subcompartment of the enzyme to another prior to degradation. This model is based primarily on the crystal structure of the proteasomal core particle (CP). The CP has a barrel-like shape, with the proteolytic active sites facing the inner chamber, or lumen. In the proteasomal CP from Thermoplasma acidophilum, openings into the lumen are found only at the ends of the barrel (59) and are therefore thought to function as channels for the proteolytic substrate. Because these channels are narrow, it is likely that proteolytic substrates must be unfolded prior to entry into the lumen of the CP. Bas...

The p44S10 locus, encoding a subunit of the proteasome regulatory particle, is amplified during progression of cutaneous malignant melanoma

“…This sequence was identical to that in Genbank Accession D14663, identi®ed previously as the human subunit 10 of the 26S proteasome regulatory particle, a multi-subunit complex that regulates core particle-mediated degradation of proteins including several oncogene or tumor suppressor gene products (Dubiel et al, 1995;Glickman et al, 1998b).…”

“…A more striking similarity occurs at residues 240 ± 348 (32% identical and 57% similar/identical), representing the PINT/PCI domain present in components of multisubunit complexes such as the proteasome, the translation initiation factor eIF3, and the cop9/ signalosome (Glickman et al, 1998a). While it remains to be determined whether up-regulation of p44 S10 expression a ects proteasome function, the expression of many growth-regulatory proteins are regulated by the proteasome pathway, including cyclins, CDK inhibitors, p53, NF-kB, c-Fos, c-Jun, and others (Hochstrasser, 1996;Coux et al, 1996;Glickman et al, 1998b). Consistent with a role for proteasome activity in tumor progression is the regulation of G1-S and M phase cell cycle transitions by ubiquitin-mediated degradation of multiple cellular proteins such as the G1 CDK inhibitor SIC1 (King et al, 1996).…”

“…The predicted polypeptides from these species were very similar in size to human p44 S10 , which contains 389 residues, and were identical in sequence in 40 ± 47% of residues. As previously shown for the human subunit, the homologous S. cerevisiae sequence was recently con®rmed as proteasome regulatory particle subunit RPN7 by denaturing gel electrophoresis and amino acid sequence analysis of the puri®ed proteasome (Dubiel et al, 1995;Glickman et al, 1998b).…”

Intracellular Dynamics of the Ubiquitin-Proteasome-System

“…The base Rpn subunits are involved in the recognition of the poly-ubiquitin chain and the Rpt ATPase subunits guide the unfolding and translocation of the polypeptide substrate into the CP ( Finley et al , 1998). In contrast to the RP base subunits, the subunits comprising the RP lid are only of the non-ATPase class: Rpn3, Rpn5-9, Rpn11 and Rpn12 ( Glickman et al , 1998). The main known function of the RP lid is the processing of poly-ubiquitin chains.…”

Genetic analysis of TAF68/61 reveals links to cell cycle regulators

“…DHH1 is a non-essential DEAD-box helicase that has been implicated in SWE1 function (Strahl-Bolsinger and Tanner, 1993;Warbick and Glover, 1994;Moriya and Isono, 1999), translation (Ladomery et al, 1997), transcription (Hata et al, 1998) and cell cycle arrest under stressful conditions (Maekawa et al, 1994;Bergkessel and Reese, unpublished). RPN11 encodes a regulatory subunit of the 26S proteasome that is required for G 2 /M progression (Glickman et al, 1998;Rinaldi et al, 1998) and for the activation of the transcription factor Gcn4p by UV-induced stress (Stitzel et al, 2001). YBR157C is the only gene product that has not been characterized; however, its expression is cell cycle-regulated, Genetic analysis of TAF68/61 1199 peaking at mitosis (Cho et al, 1998;Spellman et al, 1998).…”