Proteasomes of the yeastS. cerevisiae: genes,structure and functions

Proteasomes are large multicatalytic protease complexes which fulfil central functions in major intracellular proteolytic pathways of the eukaryotic cell. 20S proteasomes are 700 kDa cylindrically shaped particles, found in the cytoplasm and the nucleus of all eukaryotes. They are composed of a pool of 14 different subunits (MW 22–25 kDa) arranged in a stack of 4 rings with 7-fold symmetry. In the yeastSaccharomyces cerevisiae a complete set of 14 genes coding for 20S proteasome subunits have been cloned and sequenced. 26S proteasomes are even larger proteinase complexes (about 1700 kDa) which degrade ubiquitinylated proteins in an ATP-dependent fashionin vitro. The 26S proteasome is build up from the 20S proteasome as core particle and two additional 19S complexes at both ends of the 20S cylinder. Recently existence of a 26S proteasome in yeast has been demonstrated. Several 26S proteasome specific genes have been cloned and sequenced. They share similarity with a novel defined family of ATPases. 20S and 26S proteasomes are essential for functioning of the eukaryotic cell. Chromosomal deletion of 20S and 26S proteasomal genes in the yeastS. cerevisiae caused lethality of the cell. Thein vivo functions of proteasomes in major proteolytic pathways have been demonstrated by the use of 20S and 26S proteasomal mutants. Proteasomes are needed for stress dependent and ubiquitin mediated proteolysis. They are involved in the degradation of short-lived and regulatory proteins. Proteasomes are important for cell differentiation and adaptation to environmental changes. Proteasomes have also been shown to function in the control of the cell cycle.     

Processing of autophagic protein LC3 by the 20S proteasome

Ubiquitin-proteasome system and autophagy are the two major mechanisms for protein degradation in eukaryotic cells. LC3, a ubiquitin-like protein, plays an essential role in autophagy through its ability to be conjugated to phosphatidylethanolamine. In this study, we discovered a novel LC3-processing activity, and biochemically purified the 20S proteasome as the responsible enzyme. Processing of LC3 by the 20S proteasome is ATP- and ubiquitin-independent, and requires both the N-terminal helices and the ubiquitin fold of LC3; and addition of the N-terminal helices of LC3 to the N terminus of ubiquitin renders ubiquitin susceptible to 20S proteasomal activity. Further, the 20S proteasome processes LC3 in a stepwise manner, it first cleaves LC3 within its ubiquitin fold and thus disrupt the conjugation function of LC3; subsequently and especially at high concentrations of the proteasome, LC3 is completely degraded. Intriguingly, proteolysis of LC3 by the 20S proteasome can be inhibited by p62, an LC3-binding protein that mediates autophagic degradation of polyubiquitin aggregates in cells. Therefore, our study implicates a potential mechanism underlying interplay between the proteasomal and autophagic pathways. This study also provides biochemical evidence suggesting relevance of the controversial ubiquitin-independent proteolytic activity of the 20S proteasome.     

Ubiquitin-Independent Degradation of Proteins in Proteasomes

Proteasomes are large supramolecular protein complexes present in all prokaryotic and eukaryotic cells, where they perform targeted degradation of intracellular proteins. Until recently, it was generally accepted that prior to proteolytic degradation in proteasomes the proteins had to be targeted by ubiquitination: ATP-dependent attachment of (typically four sequential) residues of the low-molecular protein, ubiquitin, which involves the ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, and ubiquitin ligase. Cytoplasmic and nucleoplasmic proteins labeled in this way are then digested in 26S proteasomes. However, it becomes increasingly clear that using this route the cell eliminates only a part of unwanted proteins. Many proteins can be cleaved by the 20S proteasome in an ATP-independent manner and without previous ubiquitination. Ubiquitin-independent degradation of proteins in proteasomes is a relatively new area of studies of the role of the ubiquitin-proteasome system. However, recent data obtained in this direction already correct existing concepts about proteasomal degradation of proteins and its regulation. Ubiquitin-independent proteasome degradation needs the main structural precondition in proteins: the presence of unstructured regions in the amino acid sequences that provide interaction with the proteasome. Taking into consideration that in humans almost half of all genes encode proteins that contain a certain proportion of intrinsically disordered regions, it appears that the list of proteins undergoing ubiquitin-independent degradation will demonstrate a further increase. Since 26S proteasomes account for only 30% of the total proteasome content in mammalian cells, most of the proteasomes exist in the form of 20S complexes. The latter suggests that ubiquitin-independent proteolysis performed by the 20S proteasome is a natural process of removing damaged proteins from the cell and maintaining a constant level of intrinsically disordered proteins. In this case, the functional overload of proteasomes in aging and/or other types of pathological processes, if it is not accompanied by triggering more radical mechanisms for the elimination of damaged proteins, organelles, and whole cells, has the most serious consequences for the whole organism.     

Hybrid proteasomes. Induction by interferon-gamma and contribution to ATP-dependent proteolysis

Eukaryotic cells contain various types of proteasomes. Core 20 S proteasomes (abbreviated 20 S below) have two binding sites for the regulatory particles, PA700 and PA28. PA700-20 S-PA700 complexes are known as 26 S proteasomes and are ATP-dependent machines that degrade cell proteins. PA28 is found both in previously described complexes of the type PA28-20 S-PA28 and in complexes that also contain PA700, as PA700-20 S-PA28. We refer to the latter as "hybrid proteasomes." The relative amounts of the various types of proteasomes in HeLa extracts were determined by a combination of immunoprecipitation and immunoblotting. Hybrid proteasomes accounted for about a fourth of all proteasomes in the extracts. Association of PA28 and proteasomes proved to be ATP-dependent. Hybrid proteasomes catalyzed ATP-dependent degradation of ornithine decarboxylase (ODC) without ubiquitinylation, as do 26 S proteasomes. In contrast, the homo-PA28 complex (PA28-20 S-PA28) was incapable of degrading ODC. Intriguingly, a major immunomodulatory cytokine, interferon-gamma, appreciably enhanced the ODC degradation in HeLa and SW620 cells through induction of the hybrid proteasome, which may also be responsible for the immunological processing of intracellular antigens. Taken together, we report here for the first time the existence of two types of ATP-dependent proteases, the 26 S proteasome and the hybrid proteasome, which appear to share the ATP-dependent proteolytic pathway in mammalian cells.     

Demonstration that a human 26S proteolytic complex consists of a proteasome and multiple associated protein components and hydrolyzes ATP and ubiquitin-ligated proteins by closely linked mechanisms.

It is known that two types of high-molecular-mass protease complexes are present in the cytosol of mammalian cells; a 20S latent multicatalytic proteinase named the proteasome, and a large proteolytic complex with an apparent sedimentation coefficient of 26S that catalyzes ATP-dependent breakdown of proteins conjugated with ubiquitin. In this work, we first demonstrated that a low concentration of SDS was required for activation of the latent proteasome, whereas the 26S complex degraded substrates for proteasomes in the absence of SDS. Moreover, the 26S complex was greatly stabilized in the presence of 2 mM ATP and 20% glycerol. Based on these characteristics, we next devised a novel procedure for purification of the 26S proteolytic complexes from human kidney. In this procedure, the proteolytic complexes were precipitated from cytoplasmic extracts by ultracentrifugation for 5 h at 105000 x g, and the large 26S complexes were clearly separated from the 20S proteasomes by molecular-sieve chromatography on a Biogel A-1.5 m column. The 26S enzyme was then purified to apparent homogeneity by successive chromatographies on hydroxyapatite and Q Sepharose, then by glycerol density-gradient centrifugation. Electrophoretic and immunochemical analyses showed that the purified human 26S complex consisted of multiple subunits of proteasomes with molecular masses of 21-31 kDa and 13-15 protein components ranging in molecular mass over 35-110 kDa, which were directly associated with the proteasome. The purified 26S proteolytic complex degraded 125I-labeled lysozyme-ubiquitin conjugates in an ATP-dependent manner. The 26S enzyme also showed high ATPase activity, which was copurified with the complex. Vanadate and hemin strongly inhibited not only ATP cleavage, but also ATP-dependent breakdown of ubiquitinligated proteins, suggesting that the 26S complex hydrolyzes ATP and ubiquitinated proteins by closely linked mechanisms. These findings indicate that the 26S complex consists of a proteasome with proteolytic function and multiple other components including an ATPase that regulates energy-dependent, ubiquitin-mediated protein 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.     

Autoubiquitination of the 26S Proteasome on Rpn13 Regulates Breakdown of Ubiquitin Conjugates

Degradation rates of most proteins in eukaryotic cells are determined by their rates of ubiquitination. However, possible regulation of the proteasome's capacity to degrade ubiquitinated proteins has received little attention, although proteasome inhibitors are widely used in research and cancer treatment. We show here that mammalian 26S proteasomes have five associated ubiquitin ligases and that multiple proteasome subunits are ubiquitinated in cells, especially the ubiquitin receptor subunit, Rpn13. When proteolysis is even partially inhibited in cells or purified 26S proteasomes with various inhibitors, Rpn13 becomes extensively and selectively poly‐ubiquitinated by the proteasome‐associated ubiquitin ligase, Ube3c/Hul5. This modification also occurs in cells during heat‐shock or arsenite treatment, when poly‐ubiquitinated proteins accumulate. Rpn13 ubiquitination strongly decreases the proteasome's ability to bind and degrade ubiquitin‐conjugated proteins, but not its activity against peptide substrates. This autoinhibitory mechanism presumably evolved to prevent binding of ubiquitin conjugates to defective or stalled proteasomes, but this modification may also be useful as a biomarker indicating the presence of proteotoxic stress and reduced proteasomal capacity in cells or patients.     

Characterization of the 20S proteasome of the lepidopteran,Spodoptera frugiperda

Multiple complexes of 20S proteasomes with accessory factors play an essential role in proteolysis in eukaryotic cells. In this report, several forms of 20S proteasomes from extracts of Spodoptera frugiperda (Sf9) cells were separated using electrophoresis in a native polyacrylamide gel and examined for proteolytic activity in the gel and by Western blotting. Distinct proteasome bands isolated from the gel were subjected to liquid chromatography-tandem mass spectrometry and identified as free core particles (CP) and complexes of CP with one or two dimers of assembly chaperones PAC1-PAC2 and activators PA28γ or PA200. In contrast to the activators PA28γ and PA200 that regulate the access of protein substrates to the internal proteolytic chamber of CP in an ATP-independent manner, the 19S regulatory particle (RP) in 26S proteasomes performs stepwise substrate unfolding and opens the chamber gate in an ATP-dependent manner. Electron microscopic analysis suggested that spontaneous dissociation of RP in isolated 26S proteasomes leaves CPs with different gate sizes related presumably to different stages in the gate opening. The primary structure of 20S proteasome subunits in Sf9 cells was determined by a search of databases and by sequencing. The protein sequences were confirmed by mass spectrometry and verified by 2D gel electrophoresis. The relative rates of sequence divergence in the evolution of 20S proteasome subunits, the assembly chaperones and activators were determined by using bioinformatics. The data confirmed the conservation of regular CP subunits and PA28γ, a more accelerated evolution of PAC2 and PA200, and especially high divergence rates of PAC1.     

Chaperone-mediated 26S Proteasome Remodeling Facilitates Free K63 Ubiquitin Chain Production and Aggresome Clearance

Efficient elimination of misfolded proteins by the proteasome system is critical for proteostasis. Inadequate proteasome capacity can lead to aberrant aggregation of misfolded proteins and inclusion body formation, a hallmark of neurodegenerative disease. The proteasome system cannot degrade aggregated proteins; however, it stimulates autophagy-dependent aggregate clearance by producing unanchored lysine (K)63-linked ubiquitin chains via the proteasomal deubiquitinating enzyme Poh1. The canonical function of Poh1, which removes ubiquitin chains en bloc from proteasomal substrates prior to their degradation, requires intact 26S proteasomes. Here we present evidence that during aggresome clearance, 20S proteasomes dissociate from protein aggregates, while Poh1 and selective subunits of 19S proteasomes are retained. The dissociation of 20S proteasome components requires the molecular chaperone Hsp90. Hsp90 inhibition suppresses 26S proteasome remodeling, unanchored ubiquitin chain production, and aggresome clearance. Our results suggest that 26S proteasomes undergo active remodeling to generate a Poh1-dependent K63-deubiquitinating enzyme to facilitate protein aggregate clearance.     

Affinity Purification of the Arabidopsis 26 S Proteasome Reveals a Diverse Array of Plant Proteolytic Complexes

Selective proteolysis in plants is largely mediated by the ubiquitin (Ub)/proteasome system in which substrates, marked by the covalent attachment of Ub, are degraded by the 26 S proteasome. The 26 S proteasome is composed of two subparticles, the 20 S core protease (CP) that compartmentalizes the protease active sites and the 19 S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Here, we describe an affinity method to rapidly purify epitope-tagged 26 S proteasomes intact from Arabidopsis thaliana. In-depth mass spectrometric analyses of preparations generated from young seedlings confirmed that the 2.5-MDa CP-regulatory particle complex is actually a heterogeneous set of particles assembled with paralogous pairs for most subunits. A number of these subunits are modified post-translationally by proteolytic processing, acetylation, and/or ubiquitylation. Several proteasome-associated proteins were also identified that likely assist in complex assembly and regulation. In addition, we detected a particle consisting of the CP capped by the single subunit PA200 activator that may be involved in Ub-independent protein breakdown. Taken together, it appears that a diverse and highly dynamic population of proteasomes is assembled in plants, which may expand the target specificity and functions of intracellular proteolysis.     

Isolation and characterization of two 20S proteasomes from the endoplasmic reticulum of rat liver microsomes.

Two new forms of proteasomes, designated as the endoplasmic reticulum (ER) membrane-associated proteasome (ERa proteasome) and ER membrane-bound proteasome (ERb proteasome), were purified to homogeneity from 0.0125 and 2.5% sodium cholate extracts, respectively, of a rat liver microsomal fraction. SDS-PAGE analysis revealed that the purified ERa and ERb proteasomes were composed of multiple subunits similar to the cytosolic 20S proteasome. However, electrophoretic, structural and immunochemical differences between the ERa, ERb and cytosolic 20S proteasomes were observed on native PAGE, two-dimensional (2D) PAGE, and immunoblot analyses. Purification of ERb from a 2.5% sodium cholate extract of the trypsin-treated microsomal fraction yielded a trypsin-modified form of ERb (tERb), which lacked the C2 subunit at least. On the other hand, no ERa proteasome was obtained from the 0.0125% sodium cholate extract of the trypsin-treated microsomes, suggesting that ERa and ERb are ER membrane-associated and -bound proteasomes, respectively. The ERa, ERb, and cytosolic 20S proteasomes exhibited similar specificities as to peptide hydrolyzing activity, although differences in their activities were noted in the presence of SDS and phospholipid. With respect to the proteolysis of protein substrates, only the ERb proteasome cleaved beta-casein, and it also degraded reduced and carboxymethylated lysozyme considerably faster than the cytosolic 20S and ERa proteasomes. Collectively our results suggest that the ERa and ERb proteasomes may play roles in intracellular proteolysis distinct from that of the cytosolic 20S proteasome.     

PAN, the proteasome-activating nucleotidase from archaebacteria, is a protein-unfolding molecular chaperone

The proteasome-activating nucleotidase (PAN) from Methanococcus jannaschii is a complex of relative molecular mass 650,000 that is homologous to the ATPases in the eukaryotic 26S proteasome. When mixed with 20S archaeal proteasomes and ATP, PAN stimulates protein degradation. Here we show that PAN reduces aggregation of denatured proteins and enhances their refolding. These processes do not require ATP hydrolysis, although ATP binding enhances the ability of PAN to prevent aggregation. PAN also catalyses the unfolding of the green fluorescent protein with an 11-residue ssrA extension at its carboxy terminus (GFP11). This unfolding requires ATP hydrolysis, and is linked to GFP11 degradation when 20S proteasomes are also present. This unfolding activity seems to be essential for ATP-dependent proteolysis, although PAN may function by itself as a molecular chaperone.     

Biogenesis, structure and function of the yeast 20S proteasome.

Intracellular degradation of many eukaryotic proteins requires their covalent ligation to ubiquitin. We previously identified a ubiquitin-dependent degradation pathway in the yeast Saccharomyces cerevisiae, the DOA pathway. Independent work has suggested that a major mechanism of cellular proteolysis involves a large multisubunit protease(s) called the 20S proteasome. We demonstrate here that Doa3 and Doa5, two essential components of the DOA pathway, are subunits of the proteasome. Biochemical analyses of purified mutant proteasomes suggest functions for several conserved proteasome subunit residues. All detectable proteasome particles purified from doa3 or doa5 cells have altered physical properties; however, the mutant particles contain the same 14 different subunits as the wild-type enzyme, indicating that most or all yeast 20S proteasomes comprise a uniform population of hetero-oligomeric complexes rather than a mixture of particles of variable subunit composition. Unexpectedly, we found that the yeast Doa3 and Pre3 subunits are synthesized as precursors which are processed in a manner apparently identical to that of related mammalian proteasome subunits implicated in antigen presentation, suggesting that biogenesis of the proteasome particle is highly conserved between yeast and mammals.     

Proteasomes: protein and gene structures.

Proteasomes are ring- or cylinder-shaped particles that have a sedimentation coefficient of 20S and are composed of a characteristic set of small polypeptides. These particles have a latent multicatalytic proteinase activity. Recently, proteasomes were found to combine reversibly with multiple protein components to form 26S proteolytic complexes that catalyze ATP-dependent, selective breakdown of proteins ligated with ubiquitin. This suggests that the 26S complexes are a new type of ATP-requiring protease in eukaryotic cells. We have studied the structures of various eukaryotic proteasomes at the molecular level by physicochemical and recombinant DNA techniques and have proposed that the gross structures of proteasomes, such as their size and shape, have been highly conserved during evolution. Proteasome subunits appear to be encoded by a family of homologous genes named the "proteasome gene family," which may have evolved from a common ancestral gene. Evidence obtained by genetic analyses in yeast and studies on the levels of proteasome expression in various eukaryotic cells indicates that proteasomes have essential roles in the cell. In this review, we summarize available information on the protein and gene structures of proteasomes and discuss the biological functions of proteasomes.     

Alterations of proteasome activities in skeletal muscle tissue of diabetic rats

During the last years many investigations have shown that a major catalyst within the mechanism of skeletal muscle wasting occuring under conditions like sepsis, injuries, trauma, cancer cachexia, chronic acidosis, fasting, glucocorticoid treatment, and insulinopenia is the ubiquitin-proteasome system. Evidence for this was obtained by findings that the rate of ATP-dependent protein degradation is increased, that m-RNA concentrations of several proteasome subunits and ubiquitin are increased and the amount of ubiquitin-protein conjugates is elevated under these conditions. Additionally, the enhanced protein breakdown was shown to be suppressed by proteasome inhibitors. In the present report we show that most but not all of the proteolytic activities of partially purified 20S/26S proteasomes from skeletal muscle of rats increase after induction of Diabetes mellitus. This finding suggests that part of the mechanism of acceleration of muscle protein breakdown is due to changes in proteasome activities.     

hRpn13/ADRM1/GP110 is a novel proteasome subunit that binds the deubiquitinating enzyme, UCH37

The 26S proteasome catalyzes the degradation of most proteins in mammalian cells. To better define its composition and associated regulatory proteins, we developed affinity methods to rapidly purify 26S proteasomes from mammalian cells. By this approach, we discovered a novel 46-kDa (407 residues) subunit of its 19S regulatory complex (previously termed ADRM1 or GP110). As its N-terminal half can be incorporated into the 26S proteasome and is homologous to Rpn13, a 156-residue subunit of the 19S complex in budding yeast, we renamed it human Rpn13 (hRpn13). The C-terminal half of hRpn13 binds directly to the proteasome-associated deubiquitinating enzyme, UCH37, and enhances its isopeptidase activity. Knockdown of hRpn13 in 293T cells increases the cellular levels of ubiquitin conjugates and decreases the degradation of short-lived proteins. Surprisingly, an overproduction of hRpn13 also reduced their degradation. Furthermore, transfection of the C-terminal half of hRpn13 slows proteolysis and induces cell death, probably by acting as a dominant-negative form. Thus in human 26S proteasomes, hRpn13 appears to be important for the binding of UCH37 to the 19S complex and for efficient proteolysis.     

The proteasome-dependent proteolytic system

The 20S proteasome is an intriguingly large complex that acts as a proteolytic catalytic machine. Accumulating evidence indicates the existence of multiple factors capable of regulating the proteasome function. They are classified into two different categories, one type of regulator is PA700 or PA28 that is reversibly associated with the 20S proteasome to form enzymatically active proteasomes and the other type including a 300-kDa modulator and PI31 indirectly influences proteasome activity perhaps by promoting or suppressing the assembly of the 20S proteasome with PA700 or PA28. Thus, there have been documented two types of proteasomes composed of a core catalytic proteasome and a pair of symmetrically disposed PA700 or PA28 regulatory particle. Moreover, the recently-identified proteasome containing both PA28 and PA700 appears to play a significant role in the ATP-dependent proteolytic pathway in cells, as can the 26S proteasome which is known as a eukaryotic ATP-dependent protease.     

Extracellular, circulating proteasomes and ubiquitin - incidence and relevance

The ubiquitin-proteasome system is the major pathway for intracellular protein degradation and is also deeply involved in the regulation of most basic cellular processes. Its proteolytic core, the 20S proteasome, has found to be attached also to the cell plasma membrane and certain observations are interpreted as to suggest that they may be released into the extracellular medium, e.g. in the alveolar lining fluid, epididymal fluid and possibly during the acrosome reaction. Proteasomes have also been detected in normal human blood plasma and designated circulating proteasomes; these have a comparatively low specific activity, a distinct pattern of subtypes and their exact origin is still enigmatic. In patients suffering from autoimmune diseases, malignant myeloproliferative syndromes, multiple myeloma, acute and chronic lymphatic leukaemia, solid tumour, sepsis or trauma, respectively, the concentration of circulating proteasomes has been found to be elevated, to correlate with the disease state and has even prognostic significance. Similarly, ubiquitin has been discovered as a normal component of human blood and seminal plasma and in ovarian follicular fluid. Increased concentrations were measured in diverse pathological situations, not only in blood plasma but also in cerebrospinal fluid, where it may have neuroprotective effects. As defective spermatozoa are covered with ubiquitin in the epididymal fluid, extracellular ubiquitination is proposed to be a mechanism for quality control in spermatogenesis. Growing evidence exists also for a participation of extracellular proteasomes and ubiquitin in the fertilization process.     

26 S proteasomes function as stable entities.

Most proteins in eukaryotic cells are degraded by 26-S proteasomes, usually after being conjugated to ubiquitin. In the absence of ATP, 26-S proteasomes fall apart into their two sub-complexes, 20-S proteasomes and PA700, which reassemble upon addition of ATP. Conceivably, 26-S proteasomes dissociate and reassemble during initiation of protein degradation in a ternary complex with the substrate, as in the dissociation-reassembly cycles found for ribosomes and the chaperonin GroEL/GroES. Here we followed disassembly and assembly of 26-S proteasomes in cell extracts as the exchange of PA700 subunits between mouse and human 26-S proteasomes. Compared to the rate of proteolysis in the same extract, the disassembly-reassembly cycle was much too slow to present an obligatory step in a degradation cycle. It has been suggested that subunit S5a (Mcb1, Rpn10), which binds poly-ubiquitin substrates, shuttles between a free state and the 26-S proteasome, bringing substrate to the complex. However, S5a was not found in the free state in HeLa cells. Besides, all subunits in PA700, including S5a, exchanged at similar low rates. It therefore seems that 26-S proteasomes function as stable entities during degradation of proteins.