Discrimination between ubiquitin-dependent and ubiquitin-independent proteolytic pathways by the 26S proteasome subunit 5a.

The 26S proteasome subunit 5a binds polyubiquitin chains and has previously been shown to inhibit the degradation of mitotic cyclins. Presumably inhibition results from S5a binding and preventing recognition of Ub-cyclin conjugates by the 26S proteasome. Here we show that S5a does not inhibit the degradation of full-length ornithine decarboxylase (ODC) consistent with previous reports that the enzyme is degraded in an antizyme-dependent, but ubiquitin-independent reaction. S5a does, however, inhibit degradation of short ODC translation products generated by internal initiation events. Because in vitro translation often produces some shortened products, the existence of ubiquitin conjugated to a 35S-labeled protein is not necessarily evidence that the full-length protein is a substrate of the Ub-dependent proteolytic pathway.     

Degradation of oxidized proteins by the 20S proteasome

Oxidatively modified proteins are continuously produced in cells by reactive oxygen and nitrogen species generated as a consequence of aerobic metabolism. During periods of oxidative stress, protein oxidation is significantly increased and may become a threat to cell survival. In eucaryotic cells the proteasome has been shown (by purification of enzymatic activity, by immunoprecipitation, and by antisense oligonucleotide studies) to selectively recognize and degrade mildly oxidized proteins in the cytosol, nucleus, and endoplasmic reticulum, thus minimizing their cytotoxicity. From in vitro studies it is evident that the 20S proteasome complex actively recognizes and degrades oxidized proteins, but the 26S proteasome, even in the presence of ATP and a reconstituted functional ubiquitinylating system, is not very effective. Furthermore, relatively mild oxidative stress rapidly (but reversibly) inactivates both the ubiquitin activating/conjugating system and 26S proteasome activity in intact cells, but does not affect 20S proteasome activity. Since mild oxidative stress actually increases proteasome-dependent proteolysis (of oxidized protein substrates) the 20S 'core' proteasome complex would appear to be responsible. Finally, new experiments indicate that conditional mutational inactivation of the E1 ubiquitin-activating enzyme does not affect the degradation of oxidized proteins, further strengthening the hypothesis that oxidatively modified proteins are degraded in an ATP-independent, and ubiquitin-independent, manner by the 20S proteasome. More severe oxidative stress causes extensive protein oxidation, directly generating protein fragments, and cross-linked and aggregated proteins, that become progressively resistant to proteolytic digestion. In fact these aggregated, cross-linked, oxidized proteins actually bind to the 20S proteasome and act as irreversible inhibitors. It is proposed that aging, and various degenerative diseases, involve increased oxidative stress (largely from damaged and electron 'leaky' mitochondria), and elevated levels of protein oxidation, cross-linking, and aggregation. Since these products of severe oxidative stress inhibit the 20S proteasome, they cause a vicious cycle of progressively worsening accumulation of cytotoxic protein oxidation products.     

Tau protein degradation is catalyzed by the ATP/ubiquitin-independent 20S proteasome under normal cell conditions

Tau is the major protein exhibiting intracellular accumulation in Alzheimer disease. The mechanisms leading to its accumulation are not fully understood. It has been proposed that the proteasome is responsible for degrading tau but, since proteasomal inhibitors block both the ubiquitin-dependent 26S proteasome and the ubiqutin-independent 20S proteasome pathways, it is not clear which of these pathways is involved in tau degradation. Some involvement of the ubiquitin ligase, CHIP in tau degradation has also been postulated during stress. In the current studies, we utilized HT22 cells and tau-transfected E36 cells in order to test the relative importance or possible requirement of the ubiquitin-dependent 26S proteasomal system versus the ubiquitin-independent 20S proteasome, in tau degradation. By means of ATP-depletion, ubiquitinylation-deficient E36ts20 cells, a 19S proteasomal regulator subunit MSS1-siRNA approaches, and in vitro ubiquitinylation studies, we were able to demonstrate that ubiquitinylation is not required for normal tau degradation.     

The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover.

The 26S proteasome is an essential proteolytic complex that is responsible for degrading proteins conjugated with ubiquitin. It has been proposed that the recognition of substrates by the 26S proteasome is mediated by a multiubiquitin-chain-binding protein that has previously been characterized in both plants and animals. In this study, we identified a Saccharomyces cerevisiae homolog of this protein, designated Mcb1. Mcb1 copurified with the 26S proteasome in both conventional and nickel chelate chromatography. In addition, a significant fraction of Mcb1 in cell extracts was present in a low-molecular-mass form free of the 26S complex. Recombinant Mcb1 protein bound multiubiquitin chains in vitro and, like its plant and animal counterparts, exhibited a binding preference for longer chains. Surprisingly, (delta)mcb1 deletion mutants were viable, grew at near-wild-type rates, degraded the bulk of short-lived proteins normally, and were not sensitive to UV radiation or heat stress. These data indicate that Mcb1 is not an essential component of the ubiquitin-proteasome pathway in S.cerevisiae. However, the (delta)mcb1 mutant exhibited a modest sensitivity to amino acid analogs and had increased steady-state levels of ubiquitin-protein conjugates. Whereas the N-end rule substrate, Arg-beta-galactosidase, was degraded at the wild-type rate in the (delta)mcb1 strain, the ubiquitin fusion degradation pathway substrate, ubiquitin-Pro-beta-galactosidase, was markedly stabilized. Collectively, these data suggest that Mcb1 is not the sole factor involved in ubiquitin recognition by the 26S proteasome and that Mcb1 may interact with only a subset of ubiquitinated substrates.     

26S proteasomes and immunoproteasomes produce mainly N-extended versions of an antigenic peptide

Protein degradation by proteasomes is the source of most antigenic peptides presented on MHC class I molecules. To determine whether proteasomes generate these peptides directly or longer precursors, we developed new methods to measure the efficiency with which 26S and 20S particles, during degradation of a protein, generate the presented epitope or potential precursors. Breakdown of ovalbumin by the 26S and 20S proteasomes yielded the immunodominant peptide SIINFEKL, but produced primarily variants containing 1-7 additional N-terminal residues. Only 6-8% of the times that ovalbumin molecules were digested was a SIINFEKL or an N-extended version produced. Surprisingly, immunoproteasomes which contain the interferon-gamma-induced beta-subunits and are more efficient in antigen presentation, produced no more SIINFEKL than proteasomes. However, the immunoproteasomes released 2-4 times more of certain N-extended versions. These observations show that the changes in cleavage specificity of immunoproteasomes influence not only the C-terminus, but also the N-terminus of potential antigenic peptides, and suggest that most MHC-presented peptides result from N-terminal trimming of larger proteasome products by aminopeptidases (e.g. the interferon-gamma-induced enzyme leucine aminopeptidase).     

Impact of the N-terminal amino acid on targeted protein degradation

The N-terminus of any protein may be used as a destabilization signal for targeted protein degradation. In the eukaryotic cytosol, the signal - the so-called N-degron--is recognized for degradation by (i) the N-end rule, a well-described degradation process involving epsilon-ubiquitination; or (ii) N-terminal ubiquitination, a more recently described pathway. Dedicated E3 ubiquitin ligases known as N-recognins then act on the protein. The proteolytic pathways involve ATP-dependent chambered proteases, such as the 26S proteasome in the cytosol, which generate short oligopeptides. The N-terminus of the polypeptide chain is also important for post-proteasome degradation by specific aminopeptidases, which complete peptide cleavage to generate free amino acids. Finally, in each compartment of the eukaryotic cell, N-terminal methionine excision creates a variety of N-termini for mature proteins. It has recently been shown that the N-terminal methionine excision pathway has a major impact early in targeted protein degradation.     

A theoretical approach towards the identification of cleavage-determining amino acid motifs of the 20 S proteasome

Hitherto the mechanisms controlling the selective cleavage of peptide bonds by the 20 S proteasome have been poorly understood. The observation that peptide bond cleavage may eventually occur at the carboxyl site of either amino acid residue rules out a simple control of cleavage preferences by the P1 residue alone. Here, we follow the rationale that the presence of specific cleavage-determining amino acids motifs (CDAAMs) around the scissile peptide bond are required for the attainment of substrate conformations susceptible to cleavage. We present an exploratory search for these putative motifs based on empirical regression functions relating the cleavage probability for a given peptide bond to some selected side-chain properties of the flanking amino acid residues. Identification of the sequence locations of cleavage-determining residues relative to the scissile bond and of their optimal side-chain properties was carried out by fitting the cleavage probability to (binary) experimental observations on peptide bond cleavage gathered among a set of seven different peptide substrates with known patterns of proteolytic degradation products. In this analysis, all peptide bonds containing the same residue in the P1 position were assumed to be cleaved by the same active sites of the proteasome, and thus to be under control of the same CDAAMs. We arrived at a final set of ten different CDAAMs, accounting for the cleavage of one to five different groups of peptide bonds with an overall predictive correctness of 93 %. The CDAAM is composed of two to four "anchor" positions preferentially located between P5 and P5' around the scissile bond. This implies a length constraint for the usage of cleavage sites, which could considerably suppress the excision of shorter fragments and thus partially explain for the observed preponderance of medium-size cleavage products.     

Biochemical and physical properties of the Methanococcus jannaschii 20S proteasome and PAN, a homolog of the ATPase (Rpt) subunits of the eucaryal 26S proteasome

The 20S proteasome is a self-compartmentalized protease which degrades unfolded polypeptides and has been purified from eucaryotes, gram-positive actinomycetes, and archaea. Energy-dependent complexes, such as the 19S cap of the eucaryal 26S proteasome, are assumed to be responsible for the recognition and/or unfolding of substrate proteins which are then translocated into the central chamber of the 20S proteasome and hydrolyzed to polypeptide products of 3 to 30 residues. All archaeal genomes which have been sequenced are predicted to encode proteins with up to approximately 50% identity to the six ATPase subunits of the 19S cap. In this study, one of these archaeal homologs which has been named PAN for proteasome-activating nucleotidase was characterized from the hyperthermophile Methanococcus jannaschii. In addition, the M. jannaschii 20S proteasome was purified as a 700-kDa complex by in vitro assembly of the alpha and beta subunits and has an unusually high rate of peptide and unfolded-polypeptide hydrolysis at 100 degrees C. The 550-kDa PAN complex was required for CTP- or ATP-dependent degradation of beta-casein by archaeal 20S proteasomes. A 500-kDa complex of PAN(Delta1-73), which has a deletion of residues 1 to 73 of the deduced protein and disrupts the predicted N-terminal coiled-coil, also facilitated this energy-dependent proteolysis. However, this deletion increased the types of nucleotides hydrolyzed to include not only ATP and CTP but also ITP, GTP, TTP, and UTP. The temperature optimum for nucleotide (ATP) hydrolysis was reduced from 80 degrees C for the full-length protein to 65 degrees C for PAN(Delta1-73). Both PAN protein complexes were stable in the absence of ATP and were inhibited by N-ethylmaleimide and p-chloromercuriphenyl-sulfonic acid. Kinetic analysis reveals that the PAN protein has a relatively high V(max) for ATP and CTP hydrolysis of 3.5 and 5.8 micromol of P(i) per min per mg of protein as well as a relatively low affinity for CTP and ATP with K(m) values of 307 and 497 microM compared to other proteins of the AAA family. Based on electron micrographs, PAN and PAN(Delta1-73) apparently associate with the ends of the 20S proteasome cylinder. These results suggest that the M. jannaschii as well as related archaeal 20S proteasomes require a nucleotidase complex such as PAN to mediate the energy-dependent hydrolysis of folded-substrate proteins and that the N-terminal 73 amino acid residues of PAN are not absolutely required for this reaction.     

Growth-associated protein of 43 kDa (GAP-43) is cleaved nonprocessively by the 20S proteasome.

Purified, nonubiquitinated growth-associated protein of 43 kDa (GAP-43) was attacked by purified reticulocyte 20S proteasome but not by the 26S proteasome. Cleavage yielded 12 N-terminally labelled GAP-43 fragments that could be resolved by SDS/PAGE. Inhibitor experiments suggested that proteasome beta1 activity yielded the resolved bands and that proteasomebeta5 activity generated nonresolvable fragments. Processive degradation, yielding only nonresolvable fragments, therefore did not occur. Most of the resolved fragments co-migrated with fragments formed in the reticulocyte lysate translation mixture used for GAP-43 synthesis, which suggested that the fragments were also produced in the translation mixture by the endogenous reticulocyte lysate proteasome. Consistent with this idea, the addition of proteasome inhibitors to translation mixtures blocked fragment production. Ubiquitinated GAP-43 appeared to be the source of the fragments in the presence of ATP, and nonubiquitinated GAP-43 the source in the absence of ATP. The results therefore suggest that the lack of processing seen with the 20S proteasome is not an artefact arising from the way in which the 20S proteasome was purified. In one purification protocol, the GAP-43 fragments formed in translation mixtures co-purified with full-length GAP-43. These fragments were digested to nonresolvable products upon addition of purified 20S proteasome. Addition of calmodulin or G-actin blocked the consumption of both full-length GAP-43 and the co-purified GAP-43 fragments. This showed that the resolved fragments can re-enter the proteasome and be cleaved to nonresolvable products, indicating that the lack of processivity is not a result of their resistance to further proteasome attack. The difficult step therefore appears to be the transfer of the large fragments within the proteasome from the beta1 to the beta5 activity for further attack.     

Mammalian 26S proteasomes remain intact during protein degradation

It has been suggested that degradation of polyubiquitylated proteins is coupled to dissociation of 26S proteasomes. In contrast, using several independent types of experiments, we find that mammalian proteasomes can degrade polyubiquitylated proteins without disassembling. Thus, immobilized, (35)S-labeled 26S proteasomes degraded polyubiquitylated Sic1 and c-IAP1 without releasing any subunits. In addition, it is predicted that if 26S proteasomes dissociate into 20S proteasomes and regulatory complexes during a degradation cycle, the reassembly rate would be limiting at low proteasome concentrations. However, the rate with which each proteasome degraded polyubiquitylated Sic1 was independent of the proteasome concentration. Likewise, substrate-dependent dissociation of 26S proteasomes could not be detected by nondenaturing electrophoresis. Lastly, epoxomicin-inhibited 20S proteasomes can trap released regulatory complexes, forming inactive 26S proteasomes, but addition of epoxomicin-inhibited 20S proteasomes had no effect on the degradation of either polyubiquitylated Sic1 or UbcH10 by 26S proteasomes or of endogenous substrates in cell extracts.     

Tripeptidyl peptidase II. An oligomeric protease complex from Arabidopsis

The breakdown of most nuclear and cytoplasmic proteins involves their partial cleavage by the 26S proteasome followed by further disassembly to free amino acids by the combined action of endo- and exopeptidases. In animals, one important intermediate exopeptidase is tripeptidyl peptidase (TPP)II, which digests peptide products of the 26S proteasome and other endopeptidases into tripeptides. Here, we describe the purification and characterization of TPPII from Arabidopsis (Arabidopsis thaliana). Like its animal counterparts, Arabidopsis TPPII exists as a soluble, approximately 5- to 9-MD complex. Two related species of 153 and 142 kD are present in the purified preparations that are derived from a single TPP2 gene. Sequencing by Edman degradation of the intact polypeptides and mass spectrometry of proteolytic fragments demonstrated that the 142-kD form mainly differs from the 153-kD form by a truncation at the C-terminal end. This serine protease is a member of the subtilisin superfamily and is sensitive to the inhibitors alanine-alanine-phenylalanine-chloromethylketone and butabindide, which are diagnostic for the TPPII subfamily. The Arabidopsis TPP2 gene is widely expressed in many tissue types with related genes evident in other plant genomes. Whereas the 26S proteasome is essential, TPPII appears not as important for plant physiology. An Arabidopsis T-DNA mutant defective in TPP2 expression displays no phenotypic abnormalities and is not hypersensitive to either amino acid analogs or the 26S proteasome inhibitor MG132. As a consequence, plants likely contain other intermediate exopeptidases that assist in amino acid recycling.     

Limited degradation of oxidized calmodulin by proteasome: formation of peptides

Oxidized proteins are recognized and degraded preferentially by the proteasome. This is true for numerous proteins including calmodulin (CaM). The degradation of CaM was investigated in a human fibroblast cell line under conditions of oxidative stress. Low molecular CaM fragments or peptides were found under such conditions. In in vitro experiments it was investigated whether this CaM breakdown product formation is induced by protein oxidation or is due to a limited proteolysis-derived degradation by the 20S proteasome. Native unoxidized CaM was not degraded by 20S proteasome, oxidized CaM was degraded in a time- and H₂O₂ concentration-dependent manner. Peptides of similar molecular weight were detected in isolated calmodulin as in oxidatively stressed fibroblasts. The peptides were identified using isolated calmodulin. Therefore, in oxidatively stressed fibroblasts and in vitro CaM is forming oxidation-driven fragments and proteasomal cleavage peptides of approximately 30 amino acids which undergo a slow or no degradation.     

Differential impairment of 20S and 26S proteasome activities in human hematopoietic K562 cells during oxidative stress

The 20S proteasome and the 26S proteasome are major components of the cytosolic and nuclear proteasomal proteolytic systems. Since proteins are known to be highly susceptible targets for reactive oxygen species, the effect of H(2)O(2) treatment of K562 human hematopoietic cells toward the activities of 20S and 26S proteasomes was investigated. While the ATP-independent degradation of the fluorogenic peptide suc-LLVY-MCA was not affected by H(2)O(2) concentrations of up to 5 mM, the ATP-stimulated degradation of suc-LLVY-MCA by the 26S proteasome began to decline at 400 microM and was completely abolished at 1 mM oxidant treatment. A combination of nondenaturing electrophoresis and Western blotting let us believe that the high oxidant susceptibility of the 26S proteasome is due to oxidation of essential amino acids in the proteasome activator PA 700 which mediates the ATP-dependent proteolysis of the 26S-proteasome. The activity of the 26S-proteasome could be recovered within 24 h after exposure of cells to 1 mM H(2)O(2) but not after 2 mM H(2)O(2). In view of the specific functions of the 26S proteasome in cell cycle control and other important physiological functions, the consequences of the higher susceptibility of this protease toward oxidative stress needs to be considered.     

Selective activation of the 20 S proteasome (multicatalytic proteinase complex) by histone h3.

Two distinct activities cleaving bonds after hydrophobic amino acids have been identified in the bovine pituitary 20 S proteasome. One, expressed by the X subunit, that cleaves bonds after aromatic and branched chain amino acids was designated as chymotrypsin-like (ChT-L).(1) The second, expressed by the Y subunit, that cleaves bonds after acidic amino acids was designated as peptidylglutamyl-peptide hydrolyzing (PGPH) but also cleaves bonds after branched chain amino acids. Low micromolar concentrations of the arginine-rich histone H3 (H3) are shown to induce changes in the specificity of the proteasome by selectively activating cleavages after branched chain and acidic amino acids while inhibiting cleavage of peptidyl-arylamide bonds in synthetic substrates. H3 activates 15-fold cleavage after leucine but not phenylalanine residues in model synthetic substrates. The activation is associated with a decrease in K(m) and an increase in V(max), suggesting positive allosteric activation. H3 activates more than 60-fold degradation of the oxidized B-chain of insulin, by cleaving mainly bonds after acidic and branched chain amino acids, and accelerates the degradation of casein and lysozyme, the latter in the presence of dithiothreitol. The degradation of lysozyme in the presence of H3 generates fragments that differ from those in its absence, indicating H3-induced specificity changes. H3 inhibits cleavage of the Trp3-Ser4 and Tyr5-Gly6 bonds in gonadotropin releasing hormone, bonds cleaved by the ChT-L activity in the absence of H3. The results suggest H3-selective activation of the Y subunit and specificity changes that could potentially affect proteasomal function in the nuclear compartment.     

Role of glutaredoxin 2 and cytosolic thioredoxins in cysteinyl-based redox modification of the 20S proteasome

The yeast 20S proteasome is subject to sulfhydryl redox alterations, such as the oxidation of cysteine residues (Cys-SH) into cysteine sulfenic acid (Cys-SOH), followed by S-glutathionylation (Cys-S-SG). Proteasome S-glutathionylation promotes partial loss of chymotrypsin-like activity and post-acidic cleavage without alteration of the trypsin-like proteasomal activity. Here we show that the 20S proteasome purified from stationary-phase cells was natively S-glutathionylated. Moreover, recombinant glutaredoxin 2 removes glutathione from natively or in vitro S-glutathionylated 20S proteasome, allowing the recovery of chymotrypsin-like activity and post-acidic cleavage. Glutaredoxin 2 deglutathionylase activity was dependent on its entry into the core particle, as demonstrated by stimulating S-glutathionylated proteasome opening. Under these conditions, deglutathionylation of the 20S proteasome and glutaredoxin 2 degradation were increased when compared to non-stimulated samples. Glutaredoxin 2 fragmentation by the 20S proteasome was evaluated by SDS-PAGE and mass spectrometry, and S-glutathionylation was evaluated by either western blot analyses with anti-glutathione IgG or by spectrophotometry with the thiol reactant 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. It was also observed in vivo that glutaredoxin 2 was ubiquitinated in cellular extracts of yeast cells grown in glucose-containing medium. Other cytoplasmic oxido-reductases, namely thioredoxins 1 and 2, were also active in 20S proteasome deglutathionylation by a similar mechanism. These results indicate for the first time that 20S proteasome cysteinyl redox modification is a regulated mechanism coupled to enzymatic deglutathionylase activity.     

Molecular characterization of NbPAF encoding the alpha6 subunit of the 20S proteasome in Nicotiana benthamiana

The 26S proteasome involved in degradation of proteins covalently modified with polyubiquitin consists of the 20S proteasome and 19S regulatory complex. The NbPAF gene encoding the alpha6 subunit of the 20S proteasome was identified from Nicotiana benthamiana. NbPAF exhibits high sequence homology with the corresponding genes from Arabidopsis, human and yeast. The deduced amino acid sequence of NbPAF reveals that this protein contains the proteasome alpha-type subunits signature and nuclear localization signal at the N-terminus. The genomic Southern blot analysis suggests that the N. benthamiana genome contains one copy of NbPAF. The NbPAF mRNA was detected abundantly in flowers and weakly in roots and stems, but it was almost undetectable in mature leaves. In response to stresses, accumulation of the NbPAF mRNA was stimulated by methyl jasmonate, NaCl and salicylic acid, but not by abscisic acid and cold treatment in leaves. The NbPAF-GFP fusion protein was localized in the cytoplasm and nucleus.     

The C terminus of Rpt3, an ATPase subunit of PA700 (19 S) regulatory complex, is essential for 26 S proteasome assembly but not for activation

PA700, the 19 S regulatory subcomplex of the 26 S proteasome, contains a heterohexameric ring of AAA subunits (Rpt1 to -6) that forms the binding interface with a heteroheptameric ring of α subunits (α1 to -7) of the 20 S proteasome. Binding of these subcomplexes is mediated by interactions of C termini of certain Rpt subunits with cognate binding sites on the 20 S proteasome. Binding of two Rpt subunits (Rpt2 and Rpt5) depends on their last three residues, which share an HbYX motif (where Hb is a hydrophobic amino acid) and open substrate access gates in the center of the α ring. The relative roles of other Rpt subunits for proteasome binding and activation remain poorly understood. Here we demonstrate that the C-terminal HbYX motif of Rpt3 binds to the 20 S proteasome but does not promote proteasome gating. Binding requires the last three residues and occurs at a dedicated site on the proteasome. A C-terminal peptide of Rpt3 blocked ATP-dependent in vitro assembly of 26 S proteasome from PA700 and 20 S proteasome. In HEK293 cells, wild-type Rpt3, but not Rpt3 lacking the HbYX motif was incorporated into 26 S proteasome. These results indicate that the C terminus of Rpt3 was required for cellular assembly of this subunit into 26 S proteasome. Mutant Rpt3 was assembled into intact PA700. This result indicates that intact PA700 can be assembled independently of association with 20 S proteasome and thus may be a direct precursor for 26 S proteasome assembly under normal conditions. These results provide new insights to the non-equivalent roles of Rpt subunits in 26 S proteasome function and identify specific roles for Rpt3.     

The 26S proteasome: ubiquitin-mediated proteolysis in the tunnel

The 26S proteasome is a self-compartmentalizing protease responsible for the degradation of intracellular proteins. This giant intracellular protease is formed by several subunits arranged into two 19S polar caps-where protein recognition and ATP-dependent unfolding occur-flanking a 20S central barrel-shaped structure with an inner proteolytic chamber. Proteins targeted to the 26S proteasome are conjugated with a polyubiquitin chain by an enzymatic cascade before delivery to the 26S proteasome for degradation into oligopeptides. As a self-compartmentalizing protease, the 26S proteasome circumvents proteins not destined for degradation and can be deployed to the cytoplasmic and nuclear compartments. The 26S proteasome is a representative of emerging group of giant proteases, including tricorn protease, multicorn protease, and TPPII (tripeptidyl peptidase II).     

20S proteasome prevents aggregation of heat-denatured proteins without PA700 regulatory subcomplex like a molecular chaperone

The eukaryotic 20S proteasome is the multifunctional catalytic core of the 26S proteasome, which plays a central role in intracellular protein degradation. Association of the 20S core with a regulatory subcomplex, termed PA700 (also known as the 19S cap), forms the 26S proteasome, which degrades ubiquitinated and nonubiquitinated proteins through an ATP-dependent process. Although proteolytic assistance by this regulatory particle is a general feature of proteasome-dependent turnover, the 20S proteasome itself can degrade some proteins directly, bypassing ubiquitination and PA700, as an alternative mechanism in vitro. The mechanism underlying this pathway is based on the ability of the 20S proteasome to recognize partially unfolded proteins. Here we show that the 20S proteasome recognizes the heat-denatured forms of model proteins such as citrate synthase, malate dehydrogenase. and glyceraldehydes-3-phosphate dehydrogenase, and prevents their aggregation in vitro. This process was not followed by the refolding of these denatured substrates into their native states, whereas PA700 or the 26S proteasome generally promotes their reactivation. These results indicate that the 20S proteasome might play a role in maintaining denatured and misfolded substrates in a soluble state, thereby facilitating their refolding or degradation.     

The 26S proteasome of the fission yeast Schizosaccharomyces pombe

The 26S proteasome is the multiprotein complex that degrades proteins that have been marked for destruction by the ubiquitin pathway. It is made up of two multisubunit complexes, the 20S catalytic core and the 19S regulatory complex. We describe the isolation and characterization of conditional mutants in the regulatory complex and their use to investigate interactions between different subunits. In addition we have investigated the localization of the 26S proteasome in fission yeast, by immunofluorescence in fixed cells and live cells with the use of a GFP-tagged subunit. Surprisingly, we find that in mitotic cells the 26S proteasome occupies a discrete intracellular compartment, the nuclear periphery. Electron microscopic analysis demonstrates that the complex resides inside the nuclear envelope. During meiosis the localization showed a more dynamic distribution. In meiosis I the proteasome remained around the nuclear periphery. However, during meiosis II there was a dramatic relocalization: initially, the signal occupied the area between the dividing nuclei, but at the end of mitosis the signal dispersed, returning to the nuclear periphery on ascospore formation. This observation implies that the nuclear periphery is a major site of proteolysis in yeast during mitotic growth and raises important questions about the function of the 26S proteasome in protein degradation.