The Proteasome: a Proteolytic Nanomachine of Cell Regulation and Waste Disposal

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Biochimica et Biophysica Acta 1695 (2004) 19–31 http://www.elsevier.com/locate/bba Review The proteasome: a proteolytic nanomachine of cell regulation and waste disposal

Dieter H. Wolf *, Wolfgang Hilt

Institut fu¨r Biochemie, Universita¨t Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany

Available online 26 October 2004

Abstract

The final destination of the majority of proteins that have to be selectively degraded in eukaryotic cells is the proteasome, a highly sophisticated nanomachine essential for life. 26S proteasomes select target proteins via their modification with polyubiquitin chains or, in rare cases, by the recognition of specific motifs. They are made up of different subcomplexes, a 20S core proteasome harboring the proteolytic active sites hidden within its barrel-like structure and two 19S caps that execute regulatory functions. Similar complexes equipped with PA28 regulators instead of 19S caps are a variation of this theme specialized for the production of antigenic peptides required in immune response. Structure analysis as well as extensive biochemical and genetic studies of the 26S proteasome and the ubiquitin system led to a basic model of substrate recognition and degradation. Recent work raised new concepts. Additional factors involved in substrate acquisition and delivery to the proteasome have been discovered. Moreover, first insights in the tasks of individual subunits or subcomplexes of the 19S caps in substrate recognition and binding as well as release and recycling of polyubiquitin tags have been obtained. D 2004 Elsevier B.V. All rights reserved.

Keywords: Proteasome; Proteolysis; Ubiquitin; Regulation

1. History of cellular functions of ubiquitin-linked elimination of proteins [8–11]. The other line of research had started out The identification of the proteasome as the proteolytic with the discovery of high molecular mass particles with machine degrading those cellular proteins in vivo that had possible proteolytic activity termed cylindrical particle, been tagged with ubiquitin [1] constituted the ignition spark, prosome [12–19], alkaline protease [20] or multicatalytic which led to the explosion of knowledge of a new protease complex [21–24]. All these proteins had in regulatory principle of eukaryotic cells, now known as common that they were of cylindrical shape, had a selective proteolysis via the ubiquitin–proteasome system sedimentation constant of about 20S, were about 600 to (UPS). This system is unique in cellular regulation as—in 700 kDa in molecular mass and consisted of a variety of contrast to phosphorylation/dephosphorylation of a pro- subunits of different size. tein—it allows a complete shutdown of function of a The finding that the 20S cylinder particles and selected protein molecule due to its irreversible proteolysis. prosomes were identical with the multicatalytic protease Interestingly, this field of cellular regulation evolved from complex [25,26] led to the proposal to name this complex two different angles [2]: one line of research led to the proteasome [25]. A similar protease found in yeast, discovery of ubiquitin, its modifier function in protein proteinase yscE [27], turned out to be the equivalent of degradation and mechanistic aspects of the modification the Drosophila and mammalian proteasome [28]. The mechanism [3–7] followed by the genetic-based discovery identification of a 26S protein complex, which contained the 20S complex as a core [29–31], extended our knowledge of this proteolytic machine and laid the basis of our * Corresponding author. Tel.: +49 711 685 4390; fax: +49 711 685 4392. present understanding of many mechanistic aspects of the E-mail address: [email protected] (D.H. Wolf). now called 26S proteasome.

Discovery of an ancestor form of 20S proteasomes in the the proteasomal activity quickly uncovered several, in the archaebacterium Thermoplasma acidophilum [32] was meanwhile numerous, ubiquitin–proteasome-dependent, crucial for the structural dissection of the 20S particle. cellular processes [41,53–59]. The discovery of the central Thermoplasma 20S particles are built up of two types of importance of the proteasome in cellular regulation and the subunits, a and h. It was proposed that these two subunits lack of easy access to genetic studies in mammalian cells are the ancestors of the different a and h type subunits subsequently initiated the development of inhibitors of the gradually found in eukaryotic proteasomes [33,34]. Electron enzyme complex, which proved highly useful in mamma- microscopic studies [35,36] uncovered the basic architecture lian cell research to uncover ubiquitin–proteasome-triggered of the Thermoplasma proteasome. It consists of four rings of processes and even serve as potential therapeutic agents in seven subunits each, whereby the two outer rings are severe human diseases [60–62]. Until today, the proteasome composed of a subunits and the two inner rings of h has been found to be central and indispensable in the subunits (a7h7h7a7). The same architecture was found for regulation of a multitude of cellular processes (Fig. 1). eukaryotic proteasomes [37,38]. Molecular biological studies in yeast provided evidence that the eukaryotic proteasome contains seven different a type subunits and seven 2. The 20S core complex of the nanomachine different h type subunits [39]. Genetic studies also gave first evidence that the proteolytic activity of the proteasome The regulatory action of proteolysis in the control of cell resides in the h type subunits [1,40,41], a fact shown later functions requires a very high degree of specificity and biochemically for the Thermoplasma proteasome [42]. selectivity, not known from bordinaryQ proteases. The Electron tomographic studies laid the basis for our knowl- evolution of the eukaryotic 26S proteasome has led to a edge of the structural buildup of the 20S proteasome [43]:It new dimension in the use of proteolysis and to a proteolytic is a barrel-shaped complex with three cavities, whereby the machine, which adheres to these requirements via two two outer cavities are jointly formed by one a and one h entities: the 20S core particle [33,35,36,39,46–50,53] and subunit ring and the inner cavity is formed by the two h the 19S regulatory particle (19S cap, PA700) [63–70]. rings. Based on this intriguing structure and because the Selectivity is achieved by both entities: While the 19S cap proteasome degrades target proteins with high specificity, recognizes the substrates selectively, the catalytic 20S core the proposal was made that after unfolding degradation of particle is only able to degrade unfolded substrates, the substrate proteins occurred in the inner cavity of the excluding native, folded proteins. Selectivity and specificity proteasome barrel [44], a prediction which was later proven of the 20S core particle are achieved through the unique to be correct [45]. Elucidation of crystal structures, first of architecture of the particle: It resembles a cylinder or barrel the Thermoplasma proteasome [46] and later of the yeast with the proteolytically active sites residing in the inner core proteasome [47], was a further breakthrough leading to a of the cylinder. Such bchamberedQ protease complexes of more detailed understanding of this proteolytic machine. different composition have been found in many organisms These structural studies and simultaneous genetic studies including bacteria [71–74]. The ancestor of the eukaryotic identified the proteasome as an Ntn (N-terminal nucleo- 20S proteasome is the eubacterial and archaebacterial phile)-hydrolase with threonine as the N-terminal active proteasome. The architectural characteristic of this particle nucleophile [46–48]. They furthermore uncovered three is its composition of four seven-numbered rings, with two different h subunits conferring diverse proteolytic activities, outer rings containing a subunits and two central rings the trypsin-like, the chymotrypsin-like and the peptidyl- composed of h subunits [35,36,46,65]. Only the h subunits glutamyl-peptide cleaving (acidic) activity, to the eukaryotic carry the active sites and in bacteria each h subunit is active proteasome [47,49,50]. Additional work could define the amounting to 2Â7 identical active sites in these prokaryotic role of proteasome complexes in the degradation of proteasomes [46,48,65]. The inactive a subunits have ubiquitin-tagged proteins, which had remained an enigma evolved from the h subunits [75]. The 2Â7 a subunits for many years. In vitro studies led to the purification of two forming the two outer rings are also identical in these high molecular mass proteases of 20S and 26S whereby the prokaryotes [46,65]. In an early period in the evolution of latter species was able to degrade ubiquitinated lysozyme eukaryotes [75], the sequences of both a and h subunits of [31,51,52]. The 20S protease was shown to be actually a the 20S proteasome have diversified leading to seven subcomponent of the ATP-dependent 26S protease [30].As distinct protein subunits each (Fig. 2). Interestingly, during with the discovery of the physiological function of ubiquitin this process the number of active site h subunits has been [8], yeast genetics provided the breakthrough in the under- reduced to three yielding six active site h subunits in the standing of the in vivo function of the proteasome: A eukaryotic proteasome (Fig. 2). In parallel, the rather broad mutational defect in the chymotrypsin like activity of the amino acid specificity of the archaeal h subunits has been complex resulted in the accumulation of ubiquitinated narrowed down to three more distinct specificities, which proteins in cells, giving in vivo evidence that this proteolytic are characterized as chymotrypsin-like, trypsin-like and machine is central for the cellular degradation of ubiquitin- peptidyl-glutamyl-peptide hydrolyzing activity, the latter tagged proteins [1]. Use of yeast mutants carrying defects in cleaving after acidic amino acids [39,47,49,76]. In yeast the D.H. Wolf, W. Hilt / Biochimica et Biophysica Acta 1695 (2004) 19–31 21

Fig. 1. Cellular functions of the ubiquitin proteasome system. Regulatory protein degradation via the ubiquitin–proteasome system plays crucial roles in a large variety of essential cellular pathways. These pathways are highly interconnected, making failures in proteasomal degradation a source of severe disturbances of the cellular function network. Defects in the ubiquitin–proteasome system are the cause of severe diseases. subunits of the proteasome are encoded by 14 individual fulfill species and cell-specific roles in these organisms genes [39]. In mammals, three additional g interferon [79,80]. Elucidation of the X-ray structure of the Thermo- inducible genes exist, which encode active h-type variants plasma proteasome yielded the principles of interaction (h1i/LMP2, h2i/MECL-1 and h5i/LMP7). Incorporation of between the different subunits and identified the active site these subunits in place of the constitutive h1, h2 and h5 in the h-type subunit [46]: a and h subunits contain a subunits results in the formation of a proteasome subtype, common fold characterized by a sandwich of two h sheets the immunoproteasome, responsible for the generation of (Fig. 3). Each h sheet consists of five strands, which are antigenic peptides presented by the MHC class I complex surrounded by two a helices, H1, H2 and H3, H4 on each [77,78]. Isoforms of a and h type subunits found in side: H1 and H2 build the contacts between the a and h Drosophila and plants indicate, in addition, that besides rings, H3 and H4 mediate the interaction between h rings. proteasomes with general functions, subtypes may exist that The N-terminally located HO helix of the a subunits is

Fig. 2. Structure of the 20S core complex. The 20S proteasome is composed of a stack of four rings composed of seven subunits each. The two outer rings are made up of seven different a subunits (left graph, view from the top), whereas the two central rings are composed of seven different h subunits (right graph, view from the center). The active sites reside within the central chamber (shadow ring) of the 20S proteasome at subunits h1/Pre3, h2/Pup1 and h5/Pre2 (marked by circles). 22 D.H. Wolf, W. Hilt / Biochimica et Biophysica Acta 1695 (2004) 19–31

Fig. 3. Structure of 20S proteasome subunits of T. acidophilum. a and h subunits are shown as ribbon diagrams. Both subunits are arranged in similar orientation showing the common sandwich fold of two h sheets (green) sandwiched between two layers of a helices (red). The a subunit contains an extension at its N terminus (H0 helix). The N-terminal threonine constituting the active nucleophile in the proteolytic center of the h subunit is marked blue with the hydroxyl oxygen highlighted in orange.

replaced by a pro-sequence in the h subunits, which is X-ray studies [46,47] and genetics [48,49,76] assign cleaved off during proteasome assembly. The basic subunit proteasomes to an enzyme family designated as Ntn- fold found in the Thermoplasma proteasome is conserved in hydrolases. All active h subunits carry an N-terminal the eukaryotic proteasome [47,81]. However, eukaryotic threonine residue as nucleophile. They all are synthesized proteasome subunits contain additional structures including as precursors, which upon assembly of the particle undergo C-terminal extensions and internal loops. These moieties autolytic processing by this liberating Thr1. The three most likely determine the fixed a1 to a7 and h1 to h7 eukaryotic active h subunits are h1 (in yeast Pre3) carrying subunit arrangement. Differences found between the yeast the acidic (peptidyl-glutamyl-peptide splitting) activity, h2 [47] and the mammalian [81] subunit structures may be (in yeast Pup1) carrying the trypsin like activity and h5 (in related to the flexibility of the mammalian proteasome to yeast Pre2) harboring the chymotrypsin like activity assemble as different subtypes containing either constitutive [47,49,50,76] (Fig. 2). Proteolysis of substrates by the or inducible subunits. proteasome is processive. As found by studies on the yeast The barrel of the 20S proteasome measures 15 nm in proteasome, the length of the majority of detectable peptides length and 11 nm in diameter. The a7h7h7a7 architecture generated by proteasomal degradation is between four and leads to the formation of three chambers in the 20S fourteen amino acids [85]. Genetic studies established a particle: the catalytic chamber of about 84 nm3 formed hierarchy of the active sites in proteasomal proteolysis and by the h rings and the two antechambers formed by one their importance for cell growth: the chymotrypsin-like a and one h ring of about 59 nm3 [46,47] (Fig. 4). The activity provided by the h5 subunit is the most important, size of the antechambers indicates that they can followed by the trypsin-like activity provided by h2 and the accommodate a considerable amount of protein substrate acidic activity provided by h1 being the least important. or degradation products. The active sites in the catalytic This hierarchy is substantiated by the finding that h5h1 and chamber can only be reached by two axial pores of about h2h1 double mutants of yeast are viable, while h5h2 2 nm formed by the a subunits on each end of the double mutants are not [49,86]; for detailed review see Ref. cylindrical barrel. This pore dimension ensures that only [87]. The dominance of the chymotrypsin like activity of h5 unfolded polypeptides (single strands or double stranded for proteasomal function was made use of in numerous hairpin loops) can enter the catalytic center of the studies in mammalian cells: Inhibitors blocking this activity proteasome. In addition, access to the channel is gated: were instrumental in the discovery of proteasome functions Access to the interior is only possible after rearrangement in these cells [60,61,88]. of the N-terminal HO helices of the a subunits [82,83]. Our knowledge about the assembly of the eukaryotic 20S Opening of the gates is thought to be induced by docking proteasome structure is still limited. In contrast to the of regulatory particles, the 19S cap complexes or the archaeal proteasome where a and h rings are composed of PA28/11S activator, which is involved in proteasomal seven identical subunits, the eukaryotic 20S proteasome is antigen processing [82,84]. made up from seven different a and seven different h D.H. Wolf, W. Hilt / Biochimica et Biophysica Acta 1695 (2004) 19–31 23

Fig. 4. The ubiquitin–proteasome system. Tagging of proteasomal substrates with ubiquitin is achieved by the coordinated action of E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes and E3 ubiquitin ligases. Polyubiquitinated substrates are recognized by the 19S cap of the 26S proteasome, which can be dissociated into a lid and a base component. 19S caps are composed of 11–12 Rpn and six Rpt (ATPase) subunits. Substrates are finally degraded by the proteolytic activity of the 20S proteasome core, which is provided by three pairs of different h subunits residing in the inner chamber of the proteasome cylinder. subunits, which have to be placed in exact order. This see, Ref. [87]). Proteasomes have been found in the requires exact information in each of the subunits. The early cytoplasm, on the cytoplasmic surface of endoplasmic steps in the assembly of the eukaryotic 20S proteasome are reticulum membranes and in the nucleus of eukaryotic cells unknown. The first intermediates of mammalian [98]. Recent studies implicate that nuclear proteasomes are bhousekeepingQ proteasomes detected so far consist of one derived from precursor complexes containing Ump1 and a7 ring to which the h2, h3 and h4 subunits are attached. In unprocessed h subunits that have been imported into the case of immunoproteasomes—which are formed upon g nucleus via the classical nuclear import pathway [99]. interferon induction and, instead of the constitutive active site h subunits, contain h1i, h2i and h5i—the first intermediate found contains also a complete a7 ring and, 3. The 26S nanomachine besides h3 and h4, has h1i and h2i attached to it. In both cases, the next intermediate found is a half-proteasome with The capacity of protein selection in the cell by the one a7 ring and the h7 ring completed. This is also the first proteasome is gained through the 19S regulatory cap detectable intermediate in the assembly of yeast protea- complex, which associates with the 20S core to yield the somes. In addition, this structure contains an assembly- 26S proteasome [63,64,66]. Selection of proteasomal sub- dedicated chaperone, Ump1. Until this step the active site h strates by the 19S regulatory particle occurs via the subunits are kept in a protected and dormant state due to the recognition of polyubiquitin chains bound to proteins that presence of the covalently attached propeptide sequences. are destined to be degraded. This linkage of the 76-amino- By this means, co-translational acetylation and thus acid polypeptide ubiquitin is executed via an ATP-depend- inactivation of the N-terminal nucleophile threonine are ent mechanism by which three types of enzymes, ubiquitin prevented [86,89,90]. Additionally, the cell is protected activating enzymes E1, ubiquitin conjugating enzymes E2 from erroneous proteolysis by this mechanism. The sub- and ubiquitin-protein-ligases E3, contribute to transfer of the sequent assembly of two half-proteasomes to the mature and carboxy-terminus of the glycine76 of ubiquitin to the q- active a7h7h7a7 proteasome complex is accompanied by amino group of a lysine residue or the amino-terminus of the autocatalytic propeptide cleavage resulting in activation of selected protein to form an isopeptide or peptide bond, the three active site h subunits and followed by the digestion respectively (Fig. 4). Further addition of at least three of the assembly chaperone Ump1 [50,91–97] (for review ubiquitin moieties in line at position lysine48 of ubiquitin 24 D.H. Wolf, W. Hilt / Biochimica et Biophysica Acta 1695 (2004) 19–31 destines the protein for proteasomal degradation [8,53,100– serve as an individual binding unit of some selected 107] (Fig. 4). Interestingly, some rare cases of protein proteins. However, the ATPase ring must have major tasks selection and subsequent degradation without polyubiquitin in the transmission of the selected proteins into the tagging have been uncovered [108–110]. The 19S regu- proteolytic core of the proteasome. Therefore, it seems to latory complex (19S cap) has been intensively dissected; for be reasonable that the Rpt5 subunit tethers all selected review, see Refs. [63,64]. The 19S regulatory particle of substrates for their final preparation independent of the yeast, which is the most thoroughly studied [111],is nature of the first steps of substrate binding and delivery. composed of at least 17 different subunits. The 19S cap This hypothesis might be supported by the fact that Rpt5 can be split into two different subcomplexes, base and lid also binds the best-known non-ubiquitinated substrate of the (Fig. 4). Both subcomplexes are linked to each other via proteasome, ornithine decarboxylase, via a domain that subunit Rpn10 (S5a in mammals) (Rpn, regulatory particle imitates ubiquitin [109]. non-ATPase). The base is composed of a ring of six Recently, additional players implicated in the delivery of different ATPase subunits of the AAA-type (Rpt1 to Rpt6; ubiquitinated substrates to the proteasome have been Rpt, regulatory particle triple A protein), which dock onto elucidated. bAdaptorQ molecules bfishingQ for ubiquitinated the a rings on both ends of the 20S core and two additional substrates in the cell might be responsible for final trans- non-ATPase subunits, Rpn1 and Rpn2. The lid is built up mission of these substrate species to the proteasome holo- from eight different subunits, Rpn3 and Rpn5 to Rpn11 enzyme complex. These proteins include Dsk2, Rad23 [111] (Fig. 4). (hPlic-1, hPlic-2 and hHR23A, hHR23B in mammalian The 19S regulatory particle has to perform a set of cells) and probably Ddi1. The prominent feature of Dsk2, functions: (1) It has to recognize and bind selectively the Rad23 and Ddi1 rests in the fact that they contain two protein substrates prone to degradation; (2) these substrates different types of domains, which make them breceptorQ have to be unfolded; (3) the ubiquitin chains have to be candidates: ubiquitin-associated (UBA) and ubiquitin-like cleaved off the polyubiquitinated proteins; (4) the gates (UBL) domains. UBA domains can bind polyubiquitin and formed by the a subunits on each side of the 20S polyubiquitinated proteins; UBL domains can interact with proteasome have to be opened; and (5) the unfolded the proteasome. Indeed, a multitude of studies indicate such substrates have to be driven into the proteolytic chamber an badaptorQ or breceptorQ function for Dsk2 and Rad23 and of the 20S cylinder for degradation (Fig. 5). Our knowledge show their involvement in the degradation of polyubiquiti- of these functions is far from complete and their elucidation nated proteins in vivo [112,114,115,117–133]. Docking of is under intensive investigation. It has been shown that two Rad23 and Dsk2 onto the proteasome seems to occur at the subunits of the proteasome are able to bind polyubiquiti- base subunit Rpn1 via a leucine-rich-repeat (LRR)-like nated substrates, Rpn10 and the ATPase subunit Rpt5 domain [115,127,131]. Former studies reported a binding of [70,112–116]. As Rpn10 can be deleted in yeast without hHR23 [133] and PliC [118] to Rpn10 of the mammalian severe growth defects [116], it cannot serve as the major proteasome. Whether this reflects a difference between recognition subunit of polyubiquitinated proteins. Rpt5 is a proteasomes of yeast and mammalian cells has to be member of the ATPase ring in the 19S base. Its precise validated in future studies. Diverse adaptors might define function in the recognition process is not known yet. It may different delivery pathways to the proteasome [126,128].

Fig. 5. Mechanisms executed by the proteasome. Substrates are usually recognized via their polyubiquitin modification and bound to the 19S caps of the 26S proteasome. The substrate is then unfolded most probably by consumption of ATP via the sixfold ATPase ring residing in the 19S base and the unfolded peptide chain is transmitted to the proteolytic chamber of the 20S core (19S base and 20S proteasome are shown in a partially cut open state). During the process of transmission, the polyubiquitin tag is cleaved off by deubiquitinating enzymes. D.H. Wolf, W. Hilt / Biochimica et Biophysica Acta 1695 (2004) 19–31 25

Examples of this are the requirement of Dsk2 and Rad23 for intact polyubiquitin chains from substrates. This process is ER-associated, proteasomal degradation of a malfolded ER- ATP-dependent in vivo pointing to the fact that polyubiqui- protein, CPY*, a process which is dependent on the Cdc48– tin elimination is coupled to correct positioning of the Ufd1–Npl4 complex, but not for proteasomal degradation of substrate for cleavage. Ubp6/USP14 and Doa4/Ubp4 might a CPY* species localized to the cytoplasm, elimination of recycle ubiquitin from ubiquitin conjugates [141,148]. which is independent of the trimeric Cdc48 complex [126]. Ubp6/USP14 might function preferentially on bless Similarly, differences in the adaptor requirement for commonQ ubiquitin chains, which possibly have been proteasomal degradation of different cell cycle proteins generated by proteasome-bound ubiquitin-protein ligases were shown [128]. (E3s) as is Hul5 [138]. An interplay between proteasome- Another bdeliveryQ factor of polyubiquitinated proteins to bound E3s and ubiquitin hydrolases might tune the process the mammalian proteasome is BAC-1, an UBL domain of substrate unfolding and the speed of substrate degrada- containing protein, which in concert with CHIP, an tion. Coaction of proteasome-associated deubiquitination ubiquitin-protein-ligase, associated with the chaperone and re-polyubiquitination of a protein might furthermore Hsc70/Hsp70, targets proteins to the 26S proteasome provide a mechanism of bquality controlQ and decide [134,135] [see C. Esser et al., this journal issue]. Another between the protein’s degradation and rescue. Human example for the diversity of delivery mechanisms of UCH37, which is an enzyme cleaving ubiquitin residues proteins to the proteasome is Ufd4 (ubiquitin fusion sequentially from the distal end of a chain, might likewise degradation), an E3 ubiquitin-protein ligase, which modifies perform a bhelperQ or regulatory function in the substrate certain proteins. Binding of this E3 to the proteasome was degradation process at the proteasome [143]. found to be a prerequisite for the degradation of Ufd4 target It seems reasonable that the polyubiquitin tag of a proteins [136]. substrate protein is removed subsequent to initiation of its Matters may become even more complicated as a variety unfolding and transmission into the 20S cylinder. Due to the of additional proteasome binding proteins have been found narrow gate of the 20S proteasome, which at the most [137–139]. Cic1 of yeast, interacting with the a subunit tolerates concurrent entry of three stretches of polypeptide Pre6/a4 and binding only to fully assembled 26S protea- chains [149–151], unfolding has to be initiated prior to somes, is thought to be an adaptor protein that specifically destruction. It is thought that this function is provided by links the instable SCF-ligase components Cdc4 and Grr1 to individual ATPases Rpt1–Rpt6 of the base of the 19S this nanomachine for degradation [139]. Another prominent regulatory particle [152]. Little is known about this process proteasome binding protein is the ubiquitin-protein ligase in the eukaryotic proteasome. ATPase complexes of Hul5 (Hect ubiquitin ligase 5) of yeast [137,138]. The exact bacterial chambered proteases are able to enhance the rates function of Hul5 in the delivery of selected proteins to the of model substrate unfolding dramatically (up to 107-fold). 26S proteasome remains unclear. It may polyubiquitinate Here, signal-dependent coupling of a substrate requires the substrates that without previous ubiquitination have already binding of ATP whereas substrate denaturation needs ATP been selected. Alternatively, Hul5 may extend polyubiquitin cleavage [153–157]. The architecture of the bacterial chains of already ubiquitin-tagged substrates to tether them chambered proteases with their distal ATPase rings and tighter to the proteasome or modify existing polyubiquitin the proteolytic chamber connected through central pores is chains to modulate the recognition process or accelerate similar to the eukaryotic 26S proteasome [158–161]. This degradation [137]. architecture indicates an unfolding mechanism whereby the In addition to adaptors, in yeast and mammalian cells substrate structure is disrupted by an ATP-consuming push ubiquitin-specific proteases, UBPs, have been found asso- or pull mechanism, which drives the substrate through the ciated with the 26S proteasome. Removal of the poly- pore of the complex. After engagement of a loosely folded ubiquitin chain from the selected proteins prior to region of the substrate polypeptide chain with the protease degradation is an essential process. The process of substrate complex, multi-domain proteins are sequentially unwound deubiquitination is coupled to substrate binding and trans- by peeling off sequence elements from each structured mission. Several different deubiquitinating activities have domain as they are encountered. This process occurs in a been found that physically interact with the proteasome: highly cooperative fashion. It is clear that local, not global, Doa4/Ubp4 [140], Ubp6/USP14 [138,141], UCH37 (not protein stability determines how much work is needed for found in Saccharomyces cerevisiae) [142,143] and Rpn11/ unfolding. Denaturation and translocation of the polypeptide POH1 [144,145]. Whereas Rpn11/POH1 is an intrinsic chain are physically nonseparable events. Both operations, subunit of the 19S lid complex carrying a Zn2+ metal- unfolding and translocation, require a large amount of lopeptidase-like activity [144–147], Doa4/Ubp4, Ubp6/ energy in the form of ATP hydrolysis. The tightness of a USP14 and UCH37 are cysteine proteases, which more or local substrate domain determines the amount of ATP less transiently bind to the proteasome. These enzymes hydrolysis and consequently the speed of unfolding and show partially overlapping properties. They may provide translocation [157,162,163]. In contrast to the bacterial very specific layers of regulation to the process of chambered proteases, the eukaryotic 26S proteasome con- proteasomal substrate degradation. Rpn11/POH1 can release tains six structurally different ATPases, which seem to 26 D.H. Wolf, W. Hilt / Biochimica et Biophysica Acta 1695 (2004) 19–31 execute distinctly different functions [152]. The function of latent 20S proteasome is able to cleave natively disordered one of these, Rpt2, has been linked to the process by which substrates without the help of the 19S regulatory particle the gates of the 20S proteasome are opened by the [149]. This finding suggests that at least some disordered displacement of the N-terminal sequences of a subunits, substrates can also promote opening of the proteasome’s allowing entry of unfolded proteins into the antechamber gates. It is possible that some disordered substrates contain [82,83,152,164]. Clearly, the base of the 19S regulatory signaling features, which can target them directly for 20S particle of the proteasome harbors an inherent chaperone- proteasomal degradation without the need of polyubiquitin like activity capable to fold proteins [165]. The composition modification [149]. In this context, it is interesting that certain of the ATPase ring docking onto the 20S proteolytic hydrophobic peptides activate the proteasomal peptidase machinery is very distinct from bacterial chambered activity, most likely by influencing the gates [177]. proteases as is the rest of the regulatory particle. Never- Unfolding and transmission of a substrate is followed by theless, it seems very likely that the unfolding mechanism of its cleavage to small oligopeptide fragments. The immune the 26S proteasome is strongly similar to its bacterial system of mammalian cells makes use of certain of such counterparts [151,166,167]. Reversible association of the peptide products for antigen presentation [77,178,179] [see substrate through the polyubiquitin chain or via another also review by P. Kloetzel, this journal issue]. A regulatory targeting factor, followed by a more stable binding of a interplay of the 19S regulatory particle and the PA28/11S loosely folded region of the substrate polypeptide, might regulator, a non ATPase complex able to dock onto the 20S probably initiate mechanical unfolding. The directionality of proteasome and exerting a role in the generation of peptides a substrate’s degradation, starting from either the C- or the presented by the major histocompatibility (MHC) class I N-terminus, depends on the presence of an unstructured complex [84,180–183], might be important for the produc- region, which serves as the initiation site. Also, endopro- tion of peptides, which are well suited for antigen teolytic cleavage of natively disordered protein loops is presentation. Nevertheless, the majority of proteasome possible (Fig. 5), which explains the molecular mechanism generated peptides must be degraded further by a peptidase of the proteasomes’ ability to perfom endoproteolytic system in the cytosol [184]. Indication exists that release of cleavage and release of active transcription factors from peptides may depend on control of the 20S proteasomal inactive precursors [149,168–170] [see M. Rape and S. gates [82,164]. Jentsch, this journal issue]. Even though the last 20 years have given considerable Ubiquitinated substrates are first bound via the poly- insight into the structure, function and degradation mech- ubiquitin tag followed by unfolding at a unstructured region anism of the 26S proteasome, there is a lot more to learn in [167]. Two elements of the 19S regulatory complex are the years to come to understand this cellular nanomachine envisaged to function coordinately: The ATPase ring, which completely. Still, the different modes of delivery and grabs the substrate and through power strokes advances it binding of polyubiquitinated substrates to the 26S protea- stepwise downstream into the 20S proteasome, and an some are not well understood. Furthermore, the function of upstream site within the 19S regulatory complex, which proteasome-bound ubiquitin-protein ligases and deubiquiti- forms a restricted translocation passageway forcing reso- nating enzymes has to be defined in more detail. It has to be lution of the substrate’s protein native fold. Gly–Ala repeat clarified whether they are involved in protein selection, sequences within the Epstein–Barr virus encoded EBNA1 regulation of protein degradation or speed of degradation or protein, which most likely assume a random coil config- whether they have other tasks. In this context, it is of interest uration [171], are known to stall proteasomal degradation of to note that tight binding of a substrate via an artificially EBNA1 by this allowing immune escape of Epstein–Barr engineered linkage to the proteasome is able to circumvent virus-infected cells [172,173]. Recent studies with ornithine polyubiquitination but leads to its degradation [185], decarboxylase containing an insertion of the Gly–Ala repeat showing that linkage of substrates to the proteasome is a motif protein have given additional mechanistic insights into major task of ubiquitin chains. This finding, however, does the proteasomal degradation mechanism and have explained not exclude that the ubiquitination–deubiquitination machi- why Gly–Ala repeats block degradation of EBNA1 nery possesses additional functions in the proteasomal [174,175]. These experiments made clear that the Gly–Ala degradation process beyond substrate tethering. Further repeat acts as a stop-transfer signal in proteasome substrate unsolved questions are: What are the individual functions processing. It is postulated that the ATP motor in the of the six different ATPases Rpt1–Rpt6? What are the exact ATPase ring of the proteasome slips when it encounters mechanisms and players in the 19S regulatory particle for Gly–Ala repeats, impeding further insertion of the substrate substrate protein binding, unfolding and translocation? to the degradation center of the 20S core [174]. What are the functions of the different Rpn-subunits of Interestingly, very strongly folded protein domains, as for the 19S regulatory particle? How does gating regulate speed instance the green fluorescent protein, cannot be unfolded by of protein degradation and quality of the peptide products? the ATP motor of the proteasome [149]. In vivo unfolding can These and probably many more questions, which will be achieved by the help of the cytoplasmic chaperone appear during research on this vital cellular nanomachine, machinery [176]. It is interesting to note that the closed, will keep the scientific community busy for the next decade. D.H. Wolf, W. Hilt / Biochimica et Biophysica Acta 1695 (2004) 19–31 27

Acknowledgements [17] E. Shelton, E.L. Kuff, E.S. Maxwell, J.T. Harrington, Cytoplasmic particles and aminoacyl transferase I activity, J. Cell Biol. 45 (1970) 1–8. The authors thank Wolfgang Heinemeyer for reading the [18] J.R. Harris, The isolation and purification of a macromolecular manuscript and for helpful comments. The work of the protein component from the human erythrocyte ghost, Biochim. authors was funded by the Deutsche Forschungsgemein- Biophys. Acta 188 (1969) 31–42. schaft (DFG), Bonn, the German Federal Ministry of [19] J.R. Harris, Release of a macromolecular protein component from Education and Research within the framework of the human erythrocyte ghosts, Biochim. Biophys. Acta 150 (1968) 534–537. German-Israeli Project Cooperation (DIP), Bonn, the [20] J. Hase, K. Kobashi, N. Nakai, K. Mitsui, K. Iwata, T. Takadera, The Mukoviszidose e.V., Bonn and the Fonds der Chemischen quaternary structure of carp muscle aklaline protease, Biochim. Industrie, Frankfurt. Biophys. Acta 611 (1980) 205–213. [21] B. Dahlmann, L. Kuehn, M. Rutschmann, H. Reinauer, Purification and characterization of a multicatalytic high-molecular-mass proteinase from rat skeletal muscle, Biochem. J. 228 (1985) 161–170. References [22] S. Wilk, M. Orlowski, Evidence that pituitary cation-sensitive neutral endopeptidase is a multicatalytic protease complex, J. Neurochem. [1] W. Heinemeyer, J.A. Kleinschmidt, J. Saidowsky, C. Escher, D.H. 40 (1983) 842–849. Wolf, Proteinase yscE, the yeast proteasome/multicatalytic-multi- [23] M. Orlowski, S. Wilk, A multicatalytic protease complex from functional proteinase: mutants unravel its function in stress induced pituitary that forms enkephalin and enkephalin containing peptides, proteolysis and uncover its necessity for cell survival, EMBO J. 10 Biochem. Biophys. Res. Commun. 101 (1981) 814–822. (1991) 555–562. [24] S. Wilk, M. Orlowski, Cation-sensitive neutral endopeptidase: [2] D.H. Wolf, Proteasomes: a historical retrospective, in: W. Hilt, D.H. isolation and specificity of the bovine pituitary enzyme, J. Neuro- Wolf (Eds.), Proteasomes: The World of Regulatory Proteolysis, chem. 35 (1980) 1172–1182. Landes Bioscience, Georgetown/Eurekah.com, Austin, USA, 2000, [25] A.P. Arrigo, K. Tanaka, A.L. Goldberg, W.J. Welch, Identity of the pp. 1–7. 19S dprosome’ particle with the large multifunctional protease [3] A. Hershko, H. Heller, S. Elias, A. Ciechanover, Components of complex of mammalian cells (the proteasome), Nature 331 (1988) ubiquitin-protein ligase system. Resolution, affinity purification, and 192–194. role in protein breakdown, J. Biol. Chem. 258 (1983) 8206–8214. [26] P.E. Falkenburg, C. Haass, P.M. Kloetzel, B. Niedel, F. Kopp, L. [4] K.D. Wilkinson, M.K. Urban, A.L. Haas, Ubiquitin is the ATP- Kuehn, B. Dahlmann, Drosophila small cytoplasmic 19S ribonu- dependent proteolysis factor I of rabbit reticulocytes, J. Biol. Chem. cleoprotein is homologous to the rat multicatalytic proteinase, Nature 255 (1980) 7529–7532. 331 (1988) 190–192. [5] A. Hershko, The ubiquitin pathway for protein degradation, Trends [27] T. Achstetter, C. Ehmann, A. Osaki, D.H. Wolf, Proteolysis in Biochem. Sci. 16 (1991) 265–268. eukaryotic cells. Proteinase yscE, a new yeast peptidase, J. Biol. [6] A. Hershko, A. Ciechanover, H. Heller, A.L. Haas, I.A. Rose, Chem. 259 (1984) 13344–13348. Proposed role of ATP in protein breakdown: conjugation of protein [28] J.A. Kleinschmidt, C. Escher, D.H. Wolf, Proteinase yscE of yeast with multiple chains of the polypeptide of ATP-dependent shows homology with the 20 S cylinder particles of Xenopus laevis, proteolysis, Proc. Natl. Acad. Sci. U. S. A. 77 (1980) 1783–1786. FEBS Lett. 239 (1988) 35–40. [7] A. Ciechanover, Y. Hod, A. Hershko, A heat-stable polypeptide [29] J. Driscoll, A.L. Goldberg, The proteasome (multicatalytic protease) component of an ATP-dependent protolytic system from reticulo- is a component of the 1500-kDa proteolytic complex which cytes, Biochem. Biophys. Res. Commun. 81 (1978) 1100–1105. degrades ubiquitin-conjugated proteins, J. Biol. Chem. 265 (1990) [8] A. Varshavsky, The ubiquitin system, Trends Biochem. Sci. 22 4789–4792. (1997) 383–387. [30] E. Eytan, D. Ganoth, T. Armon, A. Hershko, ATP-dependent [9] D. Finley, E. Ozkaynak, A. Varshavsky, The yeast polyubiquitin incorporation of 20S protease into the 26S complex that degrades gene is essential for resistance to high temperatures, starvation, and proteins conjugated to ubiquitin, Proc. Natl. Acad. Sci. U. S. A. 86 other stresses, Cell 48 (1987) 1035–1046. (1989) 7751–7755. [10] A. Ciechanover, D. Finley, A. Varshavsky, Ubiquitin dependence of [31] R. Hough, G. Pratt, M. Rechsteiner, Purification of two high selective protein degradation demonstrated in the mammalian cell molecular weight proteases from rabbit reticulocyte lysate, J. Biol. cycle mutant ts85, Cell 37 (1984) 57–66. Chem. 262 (1987) 8303–8313. [11] D. Finley, A. Ciechanover, A. Varshavsky, Thermolability of [32] B. Dahlmann, F. Kopp, L. Kuehn, B. Niedel, G. Pfeifer, R. Hegerl, ubiquitin-activating enzyme from the mammalian cell cycle mutant W. Baumeister, The multicatalytic proteinase (prosome) is ubiquitous ts85, Cell 37 (1984) 43–55. from eukaryotes to archaebacteria, FEBS Lett. 251 (1989) 125–131. [12] C.V. Dang, Identity of the ubiquitous eukaryotic ring-shaped [33] P. Zwickl, A. Grziwa, G. Puhler, B. Dahlmann, F. Lottspeich, W. miniparticle, Cell Biol. Int. Rep. 8 (1984) 323–327. Baumeister, Primary structure of the Thermoplasma proteasome and [13] H.P. Schmid, O. Akhayat, C. Martins De Sa, F. Puvion, K. Koehler, its implications for the structure, function, and evolution of the K. Scherrer, The prosome: an ubiquitous morphologically distinct multicatalytic proteinase, Biochemistry 31 (1992) 964–972. RNP particle associated with repressed mRNPs and containing [34] P. Zwickl, F. Lottspeich, B. Dahlmann, W. Baumeister, Cloning and specific ScRNA and a characteristic set of proteins, EMBO J. 3 sequencing of the gene encoding the large (alpha-) subunit of the (1984) 29–34. proteasome from Thermoplasma acidophilum, FEBS Lett. 278 [14] J.A. Kleinschmidt, B. Hugle, C. Grund, W.W. Franke, The 22 (1991) 217–221. S cylinder particles of Xenopus laevis: I. Biochemical and [35] G. Pqhler, S. Weinkauf, L. Bachmann, S. Mqller, A. Engel, R. electron microscopic characterization, Eur. J. Cell Biol. 32 Hegerl, W. Baumeister, Subunit stoichiometry and three-dimensional (1983) 143–156. arrangement in proteasomes from Thermoplasma acidophilum, [15] M. Smulson, Subribosomal particles of HeLa cells, Exp. Cell Res. 87 EMBO J. 11 (1992) 1607–1616. (1974) 253–258. [36] A. Grziwa, W. Baumeister, B. Dahlmann, F. Kopp, Localization of [16] K.S. Narayan, D.E. Rounds, Minute ring-shaped particles in cultured subunits in proteasomes from Thermoplasma acidophilum by cells of malignant origin, Nat., New Biol. 243 (1973) 146–150. immunoelectron microscopy, FEBS Lett. 290 (1991) 186–190. 28 D.H. Wolf, W. Hilt / Biochimica et Biophysica Acta 1695 (2004) 19–31

[37] T.M. Schauer, M. Nesper, M. Kehl, F. Lottspeich, A. Muller- [57] B. Richter-Ruoff, D.H. Wolf, Proteasome and cell cycle. Evidence Taubenberger, G. Gerisch, W. Baumeister, Proteasomes from for a regulatory role of the protease on mitotic cyclins in yeast, FEBS Dictyostelium discoideum: characterization of structure and function, Lett. 336 (1993) 34–36. J. Struct. Biol. 111 (1993) 135–147. [58] W. Seufert, S. Jentsch, In vivo function of the proteasome in the [38] F. Kopp, B. Dahlmann, K.B. Hendil, Evidence indicating that the ubiquitin pathway, EMBO J. 11 (1992) 3077–3080. human proteasome is a complex dimer, J. Mol. Biol. 229 (1993) [59] B. Richter-Ruoff, W. Heinemeyer, D.H. Wolf, The proteasome/ 14–19. multicatalytic-multifunctional proteinase. In vivo function in the [39] W. Heinemeyer, N. Trfndle, G. Albrecht, D.H. Wolf, PRE5 ubiquitin-dependent N-end rule pathway of protein degradation in and PRE6, the last missing genes encoding 20S proteasome eukaryotes, FEBS Lett. 302 (1992) 192–196. subunits from yeast? Indication for a set of 14 different [60] D.H. Lee, A.L. Goldberg, The proteasome inhibitors and their uses, subunits in the eukaryotic proteasome core, Biochemistry 33 in: W. Hilt, D.H. Wolf (Eds.), Proteasomes: The World of Regulatory (1994) 12229–12237. Proteolysis, Landes Bioscience/Eurekah.com, Georgetown/Austin, [40] W. Heinemeyer, A. Gruhler, V. Mohrle, Y. Mahe, D.H. Wolf, PRE2, USA, 2000, pp. 154–175. highly homologous to the human major histocompatibility complex- [61] D.H. Lee, A.L. Goldberg, Proteasome inhibitors: valuable new tools linked RING10 gene, codes for a yeast proteasome subunit necessary for cell biologists, Trends Cell Biol. 8 (1998) 397–403. for chymotryptic activity and degradation of ubiquitinated proteins, [62] K.L. Rock, C. Gramm, L. Rothstein, K. Clark, R. Stein, L. Dick, D. J. Biol.Chem. 268 (1993) 5115–5120. Hwang, A.L. Goldberg, Inhibitors of the proteasome block the [41] W. Hilt, C. Enenkel, A. Gruhler, T. Singer, D.H. Wolf, The PRE4 degradation of most cell proteins and the generation of peptides gene codes for a subunit of the yeast proteasome necessary for presented on MHC class I molecules, Cell 78 (1994) 761–771. peptidylglutamyl-peptide-hydrolyzing activity. Mutations link the [63] M.H. Glickman, D.M. Rubin, C.N. Larsen, M. Schmidt, D. Finley, proteasome to stress- and ubiquitin-dependent proteolysis, J. Biol. The regulatory particle of the yeast proteasome, in: W. Hilt, D.H. Chem. 268 (1993) 3479–3486. Wolf (Eds.), Proteasomes: The World of Regulatory Proteolysis, [42] P. Zwickl, J. Kleinz, W. Baumeister, Critical elements in proteasome Landes Bioscience/Eurekah.com, Georgetown/Austin, USA, 2000, assembly, Nat. Struct. Biol. 1 (1994) 765–770. pp. 71–90. [43] R. Hegerl, G. Pfeifer, G. Puhler, B. Dahlmann, W. Baumeister, The [64] C. Gorbea, M. Rechsteiner, The mammalian regulatory complex of three-dimensional structure of proteasomes from Thermoplasma the 26S proteasome, in: W. Hilt, D.H. Wolf (Eds.), Proteasomes: The acidophilum as determined by electron microscopy using random World of Regulatory Proteolysis, Landes Bioscience, Georgetown/ conical tilting, FEBS Lett. 283 (1991) 117–121. Eurekah.com, Austin, USA, Georgetown/Austin, USA, 2000, [44] W. Hilt, D.H. Wolf, Stress-induced proteolysis in yeast, Mol. pp. 91–128. Microbiol. 6 (1992) 2437–2442. [65] W. Baumeister, J. Walz, F. Zuhl, E. Seemqller, The proteasome: [45] T. Wenzel, W. Baumeister, Conformational constraints in protein paradigm of a self-compartmentalizing protease, Cell 92 (1998) degradation by the 20s proteasome, Nat. Struct. Biol. 2 (1995) 367–380. 199–204. [66] J.M. Peters, Proteasomes: protein degradation machines of the cell, [46] J. Lfwe, D. Stock, B. Jap, P. Zwickl, W. Baumeister, R. Huber, Trends Biochem. Sci. 19 (1994) 377–382. Crystal structure of the 20S proteasome from the archaeon T. [67] M. Chu-Ping, J.H. Vu, R.J. Proske, C.A. Slaughter, G.N. DeMartino, acidophilum at 3.4 A resolution, Science 268 (1995) 533–539. Identification, purification, and characterization of a high molecular [47] M. Groll, L. Ditzel, J. Lowe, D. Stock, M. Bochtler, H.D. Bartunik, weight, ATP-dependent activator (PA700) of the 20 S proteasome, R. Huber, Structure of 20S proteasome from yeast at 2.4 A J. Biol. Chem. 269 (1994) 3539–3547. resolution, Nature 386 (1997) 463–471. [68] A. Udvardy, Purification and characterization of a multiprotein [48] E. Seemqller, A. Lupas, D. Stock, J. Lowe, R. Huber, W. Baumeister, component of the Drosophila 26 S (1500 kDa) proteolytic complex, Proteasome from Thermoplasma acidophilum: a threonine protease, J. Biol. Chem. 268 (1993) 9055–9062. Science 268 (1995) 579–582. [69] J.M. Peters, W.W. Franke, J.A. Kleinschmidt, Distinct 19 S and 20 S [49] W. Heinemeyer, M. Fischer, T. Krimmer, U. Stachon, D.H. Wolf, subcomplexes of the 26 S proteasome and their distribution in the The active sites of the eukaryotic 20 S proteasome and their nucleus and the cytoplasm, J. Biol. Chem. 269 (1994) 7709–7718. involvement in subunit precursor processing, J. Biol. Chem. 272 [70] Q. Deveraux, V. Ustrell, C. Pickart, M. Rechsteiner, A 26 S protease (1997) 25200–25209. subunit that binds ubiquitin conjugates, J. Biol. Chem. 269 (1994) [50] P. Chen, M. Hochstrasser, Autocatalytic subunit processing couples 7059–7061. active site formation in the 20S proteasome to completion of [71] M. Rohrwild, O. Coux, H.C. Huang, R.P. Moerschell, S.J. Yoo, J.H. assembly, Cell 86 (1996) 961–972. Seol, C.H. Chung, A.L. Goldberg, HslV–HslU: a novel ATP- [51] L. Waxman, J.M. Fagan, A.L. Goldberg, Demonstration of two dependent protease complex in Escherichia coli related to the distinct high molecular weight proteases in rabbit reticulocytes, one eukaryotic proteasome, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) of which degrades ubiquitin conjugates, J. Biol. Chem. 262 (1987) 5808–5813. 2451–2457. [72] S.J. Yoo, J.H. Seol, D.H. Shin, M. Rohrwild, M.S. Kang, K. [52] R. Hough, G. Pratt, M. Rechsteiner, Ubiquitin-lysozyme conju- Tanaka, A.L. Goldberg, C.H. Chung, Purification and characte- gates. Identification and characterization of an ATP-dependent rization of the heat shock proteins HslV and HslU that form a new protease from rabbit reticulocyte lysates, J. Biol. Chem. 261 (1986) ATP-dependent protease in Escherichia coli, J. Biol. Chem. 271 2400–2408. (1996) 14035–14040. [53] W. Hilt, D.H. Wolf, Proteasomes: destruction as a programme, [73] M. Kessel, W. Wu, S. Gottesman, E. Kocsis, A.C. Steven, M.R. Trends Biochem. Sci. 21 (1996) 96–102. Maurizi, Sixfold rotational symmetry of ClpQ, the E. coli homolog [54] S. Schork, G. Bee, M. Thumm, D.H. Wolf, Catabolite inactivation of of the 20S proteasome, and its ATP-dependent activator, ClpY, FEBS fructose-1,6-bisphosphatase in yeast is mediated by the proteasome, Lett. 398 (1996) 274–278. FEBS Lett. 349 (1994) 270–274. [74] D. Missiakas, F. Schwager, J.M. Betton, C. Georgopoulos, S. Raina, [55] S. Schork, G. Bee, M. Thumm, D.H. Wolf, Site of catabolite Identification and characterization of HsIV HsIU (ClpQ ClpY) inactivation, Nature 369 (1994) 283–284. proteins involved in overall proteolysis of misfolded proteins in [56] B. Richter-Ruoff, D.H. Wolf, M. Hochstrasser, Degradation of the Escherichia coli, EMBO J. 15 (1996) 6899–6909. yeast MAT alpha 2 transcriptional regulator is mediated by the [75] C. Volker, A.N. Lupas, Molecular evolution of proteasomes, Curr. proteasome, FEBS Lett. 354 (1994) 50–52. Top. Microbiol. Immunol. 268 (2002) 1–22. D.H. Wolf, W. Hilt / Biochimica et Biophysica Acta 1695 (2004) 19–31 29

[76] C.S. Arendt, M. Hochstrasser, Identification of the yeast 20S [95] D. Nandi, E. Woodward, D.B. Ginsburg, J.J. Monaco, Intermedi- proteasome catalytic centers and subunit interactions required for ates in the formation of mouse 20S proteasomes: implications for active-site formation, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) the assembly of precursor beta subunits, EMBO J. 16 (1997) 7156–7161. 5363–5375. [77] K.L. Rock, A.L. Goldberg, Degradation of cell proteins and the [96] Y. Yang, K. Fruh, K. Ahn, P.A. Peterson, In vivo assembly of the generation of MHC class I-presented peptides, Annu. Rev. Immunol. proteasomal complexes, implications for antigen processing, J. Biol. 17 (1999) 739–779. Chem. 270 (1995) 27687–27694. [78] E. Pamer, P. Cresswell, Mechanisms of MHC class I-restricted [97] S. Frentzel, B. Pesold-Hurt, A. Seelig, P.M. Kloetzel, 20 S antigen processing, Annu. Rev. Immunol. 16 (1998) 323–358. proteasomes are assembled via distinct precursor complexes. [79] H. Fu, J.H. Doelling, C.S. Arendt, M. Hochstrasser, R.D. Vierstra, Processing of LMP2 and LMP7 proproteins takes place in 13-16 S Molecular organization of the 20S proteasome gene family from preproteasome complexes, J. Mol. Biol. 236 (1994) 975–981. Arabidopsis thaliana, Genetics 149 (1998) 677–692. [98] E. Knecht, A.J. Rivett, Intracellular localization of proteasomes, in: [80] J. Ma, E. Katz, J.M. Belote, Expression of proteasome subunit W. Hilt, D.H. Wolf (Eds.), Proteasomes: The World of Regulatory isoforms during spermatogenesis in Drosophila melanogaster, Insect Proteolysis, Landes Bioscience/Eurekah.com, Georgetown/Austin, Mol. Biol. 11 (2002) 627–639. USA, 2000, pp. 176–185. [81] M. Unno, T. Mizushima, Y. Morimoto, Y. Tomisugi, K. Tanaka, N. [99] A. Lehmann, K. Janek, B. Braun, P.M. Kloetzel, C. Enenkel, 20 S Yasuoka, T. Tsukihara, The structure of the mammalian 20S proteasomes are imported as precursor complexes into the nucleus of proteasome at 2.75 A resolution, Structure (Camb) 10 (2002) yeast, J. Mol. Biol. 317 (2002) 401–413. 609–618. [100] S. Fang, A.M. Weissman, A field guide to ubiquitylation, Cell. Mol. [82] M. Bajorek, M.H. Glickman, Keepers at the final gates: regulatory Life Sci. 61 (2004) 1546–1561. complexes and gating of the proteasome channel, Cell. Mol. Life Sci. [101] A. Ciechanover, R. Ben-Saadon, N-terminal ubiquitination: more 61 (2004) 1579–1588. protein substrates join in, Trends Cell Biol. 14 (2004) 103–106. [83] M. Groll, M. Bajorek, A. Kohler, L. Moroder, D.M. Rubin, R. [102] K. Breitschopf, E. Bengal, T. Ziv, A. Admon, A. Ciechanover, A Huber, M.H. Glickman, D. Finley, A gated channel into the novel site for ubiquitination: the N-terminal residue, and not internal proteasome core particle, Nat. Struct. Biol. 7 (2000) 1062–1067. lysines of MyoD, is essential for conjugation and degradation of the [84] C.P. Hill, E.I. Masters, F.G. Whitby, The 11S regulators of 20S protein, EMBO J. 17 (1998) 5964–5973. proteasome activity, Curr. Top. Microbiol. Immunol. 268 (2002) [103] C.M. Pickart, Mechanisms underlying ubiquitination, Annu. Rev. 73–89. Biochem. 70 (2001) 503–533. [85] A.K. Nussbaum, T.P. Dick, W. Keilholz, M. Schirle, S. Stevanovic, [104] J.S. Thrower, L. Hoffman, M. Rechsteiner, C.M. Pickart, Recog- K. Dietz, W. Heinemeyer, M. Groll, D.H. Wolf, R. Huber, H.G. nition of the polyubiquitin proteolytic signal, EMBO J. 19 (2000) Rammensee, H. Schild, Cleavage motifs of the yeast 20S proteasome 94–102. beta subunits deduced from digests of enolase 1, Proc. Natl. Acad. [105] C.M. Pickart, Ubiquitin in chains, Trends Biochem. Sci. 25 (2000) Sci. U. S. A. 95 (1998) 12504–12509. 544–548. [86] S. J7ger, M. Groll, R. Huber, D.H. Wolf, W. Heinemeyer, [106] A. Hershko, A. Ciechanover, The ubiquitin system, Annu. Rev. Proteasome beta-type subunits: unequal roles of propeptides in core Biochem. 67 (1998) 425–479. particle maturation and a hierarchy of active site function, J. Mol. [107] V. Chau, J.W. Tobias, A. Bachmair, D. Marriott, D.J. Ecker, D.K. Biol. 291 (1999) 997–1013. Gonda, A. Varshavsky, A multiubiquitin chain is confined to [87] W. Heinemeyer, P.C. Ramos, R.J. Dohmen, The ultimate nanoscale specific lysine in a targeted short-lived protein, Science 243 mincer: assembly, structure and active sites of the 20S proteasome (1989) 1576–1583. core, Cell. Mol. Life Sci. 61 (2004) 1562–1578. [108] M.A. Hoyt, P. Coffino, Ubiquitin-free routes into the proteasome, [88] M. Bogyo, E.W. Wang, Proteasome inhibitors: complex tools for a Cell. Mol. Life Sci. 61 (2004) 1596–1600. complex enzyme, Curr. Top. Microbiol. Immunol. 268 (2002) [109] M. Zhang, C.M. Pickart, P. Coffino, Determinants of proteasome 185–208. recognition of ornithine decarboxylase, a ubiquitin-independent [89] M. Groll, W. Heinemeyer, S. Jager, T. Ullrich, M. Bochtler, D.H. substrate, EMBO J. 22 (2003) 1488–1496. Wolf, R. Huber, The catalytic sites of 20S proteasomes and their role [110] P. Coffino, Regulation of cellular polyamines by antizyme, Nat. Rev., in subunit maturation: a mutational and crystallographic study, Proc. Mol. Cell Biol. 2 (2001) 188–194. Natl. Acad. Sci. U. S. A. 96 (1999) 10976–10983. [111] M.H. Glickman, D.M. Rubin, O. Coux, I. Wefes, G. Pfeifer, Z. [90] C.S. Arendt, M. Hochstrasser, Eukaryotic 20S proteasome catalytic Cjeka, W. Baumeister, V.A. Fried, D. Finley, A subcomplex of the subunit propeptides prevent active site inactivation by N-terminal proteasome regulatory particle required for ubiquitin-conjugate acetylation and promote particle assembly, EMBO J. 18 (1999) degradation and related to the COP9-signalosome and eIF3, Cell 3575–3585. 94 (1998) 615–623. [91] E. Witt, D. Zantopf, M. Schmidt, R. Kraft, P.M. Kloetzel, E. Kruger, [112] S. Elsasser, D. Chandler-Militello, B. Muller, J. Hanna, D. Finley, Characterisation of the newly identified human Ump1 homologue Rad23 and Rpn10 serve as alternative ubiquitin receptors for the POMP and analysis of LMP7(beta 5i) incorporation into 20 S proteasome, J. Biol. Chem. 279 (2004) 26817–26822. proteasomes, J. Mol. Biol. 301 (2000) 1–9. [113] Y.A. Lam, T.G. Lawson, M. Velayutham, J.L. Zweier, C.M. Pickart, [92] T.A. Griffin, J.P. Slack, T.S. McCluskey, J.J. Monaco, R.A. Colbert, A proteasomal ATPase subunit recognizes the polyubiquitin degra- Identification of proteassemblin, a mammalian homologue of the dation signal, Nature 416 (2002) 763–767. yeast protein, Ump1p, that is required for normal proteasome [114] R. Hartmann-Petersen, M. Seeger, C. Gordon, Transferring assembly, Mol. Cell. Biol. Res. Commun. 3 (2000) 212–217. substrates to the 26S proteasome, Trends Biochem. Sci. 28 [93] L. Burri, J. Hockendorff, U. Boehm, T. Klamp, R.J. Dohmen, F. (2003) 26–31. Levy, Identification and characterization of a mammalian protein [115] S. Elsasser, R.R. Gali, M. Schwickart, C.N. Larsen, D.S. Leggett, B. interacting with 20S proteasome precursors, Proc. Natl. Acad. Sci. Muller, M.T. Feng, F. Tubing, G.A. Dittmar, D. Finley, Proteasome U. S. A. 97 (2000) 10348–10353. subunit Rpn1 binds ubiquitin-like protein domains, Nat. Cell Biol. 4 [94] P.C. Ramos, J. Hockendorff, E.S. Johnson, A. Varshavsky, R.J. (2002) 725–730. Dohmen, Ump1p is required for proper maturation of the 20S [116] S. van Nocker, S. Sadis, D.M. Rubin, M. Glickman, H. Fu, O. Coux, proteasome and becomes its substrate upon completion of the I. Wefes, D. Finley, R.D. Vierstra, The multiubiquitin-chain-binding assembly, Cell 92 (1998) 489–499. protein Mcb1 is a component of the 26S proteasome in Saccha- 30 D.H. Wolf, W. Hilt / Biochimica et Biophysica Acta 1695 (2004) 19–31

romyces cerevisiae and plays a nonessential, substrate-specific role [138] D.S. Leggett, J. Hanna, A. Borodovsky, B. Crosas, M. Schmidt, R.T. in protein turnover, Mol. Cell. Biol. 16 (1996) 6020–6028. Baker, T. Walz, H. Ploegh, D. Finley, Multiple associated proteins [117] T.G. Ortolan, P. Tongaonkar, D. Lambertson, L. Chen, C. Schauber, regulate proteasome structure and function, Mol. Cell 10 (2002) K. Madura, The DNA repair protein rad23 is a negative regulator of 495–507. multi-ubiquitin chain assembly, Nat. Cell Biol. 2 (2000) 601–608. [139] S. J7ger, J. Strayle, W. Heinemeyer, D.H. Wolf, Cic1, an adaptor [118] K.J. Walters, M.F. Kleijnen, A.M. Goh, G. Wagner, P.M. Howley, protein specifically linking the 26S proteasome to its substrate, the Structural studies of the interaction between ubiquitin family proteins SCF component Cdc4, EMBO J. 20 (2001) 4423–4431. and proteasome subunit S5a, Biochemistry 41 (2002) 1767–1777. [140] F.R. Papa, A.Y. Amerik, M. Hochstrasser, Interaction of the Doa4 [119] R. Hartmann-Petersen, C. Gordon, Proteins interacting with the 26S deubiquitinating enzyme with the yeast 26S proteasome, Mol. Biol. proteasome, Cell. Mol. Life Sci. 61 (2004) 1589–1595. Cell 10 (1999) 741–756. [120] L. Chen, U. Shinde, T.G. Ortolan, K. Madura, Ubiquitin-associated [141] A. Borodovsky, B.M. Kessler, R. Casagrande, H.S. Overkleeft, K.D. (UBA) domains in Rad23 bind ubiquitin and promote inhibition of Wilkinson, H.L. Ploegh, A novel active site-directed probe specific multi-ubiquitin chain assembly, EMBO Rep. 2 (2001) 933–938. for deubiquitylating enzymes reveals proteasome association of [121] C. Schauber, L. Chen, P. Tongaonkar, I. Vega, D. Lambertson, W. USP14, EMBO J. 20 (2001) 5187–5196. Potts, K. Madura, Rad23 links DNA repair to the ubiquitin/ [142] H. Hflzl, B. Kapelari, J. Kellermann, E. Seemuller, M. Sumegi, A. proteasome pathway, Nature 391 (1998) 715–718. Udvardy, O. Medalia, J. Sperling, S.A. Muller, A. Engel, W. [122] H. Rao, A. Sastry, Recognition of specific ubiquitin conjugates is Baumeister, The regulatory complex of Drosophila melanogaster important for the proteolytic functions of the ubiquitin-associated 26S proteasomes. Subunit composition and localization of a domain proteins Dsk2 and Rad23, J. Biol. Chem. 277 (2002) deubiquitylating enzyme, J. Cell Biol. 150 (2000) 119–130. 11691–11695. [143] Y.A. Lam, W. Xu, G.N. DeMartino, R.E. Cohen, Editing of ubiquitin [123] M.F. Kleijnen, A.H. Shih, P. Zhou, S. Kumar, R.E. Soccio, N.L. conjugates by an isopeptidase in the 26S proteasome, Nature 385 Kedersha, G. Gill, P.M. Howley, The hPLIC proteins may provide a (1997) 737–740. link between the ubiquitination machinery and the proteasome, Mol. [144] T. Yao, R.E. Cohen, A cryptic protease couples deubiquitination and Cell 6 (2000) 409–419. degradation by the proteasome, Nature 419 (2002) 403–407. [124] M. Funakoshi, T. Sasaki, T. Nishimoto, H. Kobayashi, Budding yeast [145] R. Verma, L. Aravind, R. Oania, W.H. McDonald, J.R. Yates III, E.V. Dsk2p is a polyubiquitin-binding protein that can interact with the Koonin, R.J. Deshaies, Role of Rpn11 metalloprotease in deubiqui- proteasome, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 745–750. tination and degradation by the 26S proteasome, Science 298 (2002) [125] S. Glockzin, F.X. Ogi, A. Hengstermann, M. Scheffner, C. Blattner, 611–615. Involvement of the DNA repair protein hHR23 in p53 degradation, [146] M.H. Glickman, N. Adir, The proteasome and the delicate balance Mol. Cell. Biol. 23 (2003) 8960–8969. between destruction and rescue, PLoS Biol. 2 (2004) E13. [126] B. Medicherla, Z. Kostova, A. Schaefer, D.H. Wolf, A genomic [147] V. Maytal-Kivity, N. Reis, K. Hofmann, M.H. Glickman, MPN+, a screen identifies Dsk2p and Rad23p as essential components of ER- putative catalytic motif found in a subset of MPN domain proteins associated degradation, EMBO Rep. 5 (2004) 692–697. from eukaryotes and prokaryotes, is critical for Rpn11 function, [127] I. Kim, K. Mi, H. Rao, Multiple interactions of rad23 suggest a BMC Biochem. 3 (2002) 28. mechanism for ubiquitylated substrate delivery important in proteo- [148] A.Y. Amerik, J. Nowak, S. Swaminathan, M. Hochstrasser, The lysis, Mol. Biol. Cell 15 (2004) 3357–3365. Doa4 deubiquitinating enzyme is functionally linked to the vacuolar [128] R. Verma, R. Oania, J. Graumann, R.J. Deshaies, Multiubiquitin protein-sorting and endocytic pathways, Mol. Biol. Cell 11 (2000) chain receptors define a layer of substrate selectivity in the ubiquitin– 3365–3380. proteasome system, Cell 118 (2004) 99–110. [149] C.W. Liu, M.J. Corboy, G.N. DeMartino, P.J. Thomas, Endoproteo- [129] Y. Saeki, A. Saitoh, A. Toh-e, H. Yokosawa, Ubiquitin-like proteins lytic activity of the proteasome, Science 299 (2003) 408–411. and Rpn10 play cooperative roles in ubiquitin-dependent proteolysis, [150] M. Rape, S. Jentsch, Taking a bite: proteasomal protein processing, Biochem. Biophys. Res. Commun. 293 (2002) 986–992. Nat. Cell Biol. 4 (2002) E113–E116. [130] L. Chen, K. Madura, Rad23 promotes the targeting of proteolytic [151] C. Lee, S. Prakash, A. Matouschek, Concurrent translocation of substrates to the proteasome, Mol. Cell. Biol. 22 (2002) 4902–4913. multiple polypeptide chains through the proteasomal degradation [131] Y. Saeki, T. Sone, A. Toh-e, H. Yokosawa, Identification of channel, J. Biol. Chem. 277 (2002) 34760–34765. ubiquitin-like protein-binding subunits of the 26S proteasome, [152] D.M. Rubin, M.H. Glickman, C.N. Larsen, S. Dhruvakumar, D. Biochem. Biophys. Res. Commun. 296 (2002) 813–819. Finley, Active site mutants in the six regulatory particle ATPases [132] C.R. Wilkinson, M. Seeger, R. Hartmann-Petersen, M. Stone, M. reveal multiple roles for ATP in the proteasome, EMBO J. 17 (1998) Wallace, C. Semple, C. Gordon, Proteins containing the UBA 4909–4919. domain are able to bind to multi-ubiquitin chains, Nat. Cell Biol. 3 [153] Y.I. Kim, R.E. Burton, B.M. Burton, R.T. Sauer, T.A. Baker, (2001) 939–943. Dynamics of substrate denaturation and translocation by the ClpXP [133] H. Hiyama, M. Yokoi, C. Masutani, K. Sugasawa, T. Maekawa, K. degradation machine, Mol. Cell 5 (2000) 639–648. Tanaka, J.H. Hoeijmakers, F. Hanaoka, Interaction of hHR23 with [154] S.K. Singh, R. Grimaud, J.R. Hoskins, S. Wickner, M.R. Maurizi, S5a. The ubiquitin-like domain of hHR23 mediates interaction with Unfolding and internalization of proteins by the ATP-dependent S5a subunit of 26 S proteasome, J. Biol. Chem. 274 (1999) proteases ClpXP and ClpAP, Proc. Natl. Acad. Sci. U. S. A. 97 28019–28025. (2000) 8898–8903. [134] D.M. Cyr, J. Hfhfeld, C. Patterson, Protein quality control: U-box- [155] J.R. Hoskins, S.K. Singh, M.R. Maurizi, S. Wickner, Protein containing E3 ubiquitin ligases join the fold, Trends Biochem. Sci. binding and unfolding by the chaperone ClpA and degradation by 27 (2002) 368–375. the protease ClpAP, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) [135] J. Hfhfeld, D.M. Cyr, C. Patterson, From the cradle to the grave: 8892–8897. molecular chaperones that may choose between folding and [156] E.U. Weber-Ban, B.G. Reid, A.D. Miranker, A.L. Horwich, Global degradation, EMBO Rep. 2 (2001) 885–890. unfolding of a substrate protein by the Hsp100 chaperone ClpA, [136] Y. Xie, A. Varshavsky, UFD4 lacking the proteasome-binding region Nature 401 (1999) 90–93. catalyses ubiquitination but is impaired in proteolysis, Nat. Cell Biol. [157] R.E. Burton, S.M. Siddiqui, Y.I. Kim, T.A. Baker, R.T. Sauer, Effects 4 (2002) 1003–1007. of protein stability and structure on substrate processing by the [137] J. You, C.M. Pickart, A HECT domain E3 enzyme assembles novel ClpXP unfolding and degradation machine, EMBO J. 20 (2001) polyubiquitin chains, J. Biol. Chem. 276 (2001) 19871–19878. 3092–3100. D.H. Wolf, W. Hilt / Biochimica et Biophysica Acta 1695 (2004) 19–31 31

[158] F. Guo, M.R. Maurizi, L. Esser, D. Xia, Crystal structure of ClpA, an [172] J. Levitskaya, A. Sharipo, A. Leonchiks, A. Ciechanover, M.G. Hsp100 chaperone and regulator of ClpAP protease, J. Biol. Chem. Masucci, Inhibition of ubiquitin/proteasome-dependent protein 277 (2002) 46743–46752. degradation by the Gly–Ala repeat domain of the Epstein–Barr [159] J. Wang, J.J. Song, M.C. Franklin, S. Kamtekar, Y.J. Im, S.H. Rho, virus nuclear antigen 1, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) I.S. Seong, C.S. Lee, C.H. Chung, S.H. Eom, Crystal structures of 12616–12621. the HslVU peptidase–ATPase complex reveal an ATP-dependent [173] J. Levitskaya, M. Coram, V. Levitsky, S. Imreh, P.M. Steigerwald- proteolysis mechanism, Structure (Camb) 9 (2001) 177–184. Mullen, G. Klein, M.G. Kurilla, M.G. Masucci, Inhibition of antigen [160] M.C. Sousa, C.B. Trame, H. Tsuruta, S.M. Wilbanks, V.S. Reddy, processing by the internal repeat region of the Epstein-Barr virus D.B. McKay, Crystal and solution structures of an HslUV protease– nuclear antigen-1, Nature 375 (1995) 685–688. chaperone complex, Cell 103 (2000) 633–643. [174] M. Zhang, P. Coffino, Repeat sequence of Epstein–Barr virus- [161] P. Zwickl, D. Ng, K.M. Woo, H.P. Klenk, A.L. Goldberg, An encoded nuclear antigen 1 protein interrupts proteasome substrate archaebacterial ATPase, homologous to ATPases in the eukaryotic processing, J. Biol. Chem. 279 (2004) 8635–8641. 26 S proteasome, activates protein breakdown by 20 S proteasomes, [175] M. Zhang, A.I. MacDonald, M.A. Hoyt, P. Coffino, Proteasomes J. Biol. Chem. 274 (1999) 26008–26014. begin ornithine decarboxylase digestion at the C terminus, J. Biol. [162] J.A. Kenniston, T.A. Baker, J.M. Fernandez, R.T. Sauer, Linkage Chem. 279 (2004) 20959–20965. between ATP consumption and mechanical unfolding during the [176] C. Taxis, R. Hitt, S.H. Park, P.M. Deak, Z. Kostova, D.H. Wolf, Use protein processing reactions of an AAA+ degradation machine, Cell of modular substrates demonstrates mechanistic diversity and reveals 114 (2003) 511–520. differences in chaperone requirement of ERAD, J. Biol. Chem. 278 [163] C. Lee, M.P. Schwartz, S. Prakash, M. Iwakura, A. Matouschek, (2003) 35903–35913. ATP-dependent proteases degrade their substrates by processively [177] A.F. Kisselev, D. Kaganovich, A.L. Goldberg, Binding of hydro- unraveling them from the degradation signal, Mol. Cell 7 (2001) phobic peptides to several non-catalytic sites promotes peptide 627–637. hydrolysis by all active sites of 20 S proteasomes. Evidence for [164] A. Koehler, P. Cascio, D.S. Leggett, K.M. Woo, A.L. Goldberg, D. peptide-induced channel opening in the alpha-rings, J. Biol. Chem Finley, The axial channel of the proteasome core particle is gated by 277 (2002) 22260–22270. the Rpt2 ATPase and controls both substrate entry and product [178] P.M. Kloetzel, Antigen processing by the proteasome, Nat. Rev., release, Mol. Cell 7 (2001) 1143–1152. Mol. Cell Biol. 2 (2001) 179–187. [165] B.C. Braun, M. Glickman, R. Kraft, B. Dahlmann, P.M. Kloetzel, D. [179] K.L. Rock, I.A. York, A.L. Goldberg, Post-proteasomal antigen Finley, M. Schmidt, The base of the proteasome regulatory particle processing for major histocompatibility complex class I presentation, exhibits chaperone-like activity, Nat. Cell Biol. 1 (1999) 221–226. Nat. Immunol. 5 (2004) 670–677. [166] A. Matouschek, Protein unfolding—an important process in vivo? [180] F.G. Whitby, E.I. Masters, L. Kramer, J.R. Knowlton, Y. Yao, C.C. Curr. Opin. Struct. Biol. 13 (2003) 98–109. Wang, C.P. Hill, Structural basis for the activation of 20S [167] S. Prakash, L. Tian, K.S. Ratliff, R.E. Lehotzky, A. Matouschek, An proteasomes by 11S regulators, Nature 408 (2000) 115–120. unstructured initiation site is required for efficient proteasome- [181] W. Dubiel, G. Pratt, K. Ferrell, M. Rechsteiner, Purification of an 11 mediated degradation, Nat. Struct. Mol. Biol. 11 (2004) 830–837. S regulator of the multicatalytic protease, J. Biol. Chem. 267 (1992) [168] L. Lin, G.N. DeMartino, W.C. Greene, Cotranslational biogenesis of 22369–22377. NF-kappaB p50 by the 26S proteasome, Cell 92 (1998) 819–828. [182] C.P. Ma, C.A. Slaughter, G.N. DeMartino, Identification, purifica- [169] M. Rape, T. Hoppe, I. Gorr, M. Kalocay, H. Richly, S. Jentsch, tion, and characterization of a protein activator (PA28) of the 20 S Mobilization of processed, membrane-tethered SPT23 transcription proteasome (macropain), J. Biol. Chem. 267 (1992) 10515–10523. factor by CDC48(UFD1/NPL4), a ubiquitin-selective chaperone, [183] W. Dubiel, P.M. Kloetzel, The 20S proteasome activator PA28 or Cell 107 (2001) 667–677. 11S regulator, in: W. Hilt, D.H. Wolf (Eds.), Proteasomes: The World [170] T. Hoppe, K. Matuschewski, M. Rape, S. Schlenker, H.D. Ulrich, S. of Regulatory Proteolysis, Landes Bioscience/Eurekah.com, George- Jentsch, Activation of a membrane-bound transcription factor by town/Austin, USA, 2000, pp. 129–135. regulated ubiquitin/proteasome-dependent processing, Cell 102 [184] M. Bqchler, U. Tisljar, D.H. Wolf, Proteinase yscD (oligopeptidase (2000) 577–586. yscD). Structure, function and relationship of the yeast enzyme with [171] A. Leonchiks, E. Liepinsh, M. Barishev, A. Sharipo, M.G. Masucci, mammalian thimet oligopeptidase (metalloendopeptidase, EP 24.15), G. Otting, Random coil conformation of a Gly/Ala-rich insert in Eur. J. Biochem. 219 (1994) 627–639. IkappaB alpha excludes structural stabilization as the mechanism for [185] D.M. Janse, B. Crosas, D. Finley, G.M. Church, Localization to the protection against proteasomal degradation, FEBS Lett. 440 (1998) proteasome is sufficient for degradation, J. Biol. Chem. 279 (2004) 365–369. 21415–21420.