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Dis-assembly lines: the proteasome and related ATPase-assisted proteases
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Dis-assembly lines: the proteasome and related ATPase-assisted proteases Peter Zwickl*, Wolfgang Baumeister*† and Alasdair Steven‡ Self-compartmentalizing proteases, such as the proteasome and several prokaryotic energy-dependent proteases, are designed to act in the crowded environment of the cell. Proteins destined for degradation are recognized and unfolded by regulatory subcomplexes that invariably contain ATPase modules, before being translocated into another subcomplex, the proteolytic core, for degradation. The sequential actions effected on substrates are reflected in the linear arrangement of these subcomplexes; thus, the holocomplexes are organized as molecular disassembly and degradation lines.
to obtain homogeneous preparations. This lability is attributable primarily to the 19S component, whose complexity — it is built of at least 17 distinct subunits — poses a further challenge, exacerbated by its lack of symmetry. In this review, we summarize some recent advances that deepen our understanding of the structure, assembly and function of the 20S proteasome, but the main emphasis is on properties of the 19S particle. We also develop a comparison between proteasomes and simpler, but functionally related, complexes of bacterial descent.
Addresses *Department of Molecular Structural Biology, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany † e-mail: [email protected] ‡ Laboratory of Structural Biology, Building 6, Room B2-34, Center Drive, MSC-2717, NIAMS, National Institutes of Health, Bethesda, MD 20892-2717, USA
20S proteasomes
Current Opinion in Structural Biology 2000, 10:242–250 0959-440X/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations AAA ATPases associated with a variety of cellular activities ARC ATPase forming ring-shaped complexes Clp caseinolytic protease NSF N-ethylmaleimide sensitive factor ODC ornithine decarboxylase PAN proteasome activating nucleotidase SSD sensor and substrate discrimination ssrA chromosomal 10Sa gene VAT valosine-containing protein-like ATPase from Thermoplasma acidophilum
Introduction In eukaryotes, the 26S proteasome is the central protease in the energy-dependent degradation of proteins via the ubiquitin pathway. This quintessential molecular machine, with a cumulative mass of more than 2.5 MDa, comprises two subcomplexes — the 20S core particle and the 19S regulatory particle. The core particle performs the actual proteolysis, whereas the 19S particle recognizes and unfolds substrates and delivers them for degradation [1]. These activities are coordinated in a manner reminiscent of an industrial assembly line acting in reverse: in this case, the starting material — a protein targeted for degradation — is a complex entity that is sequentially dismantled, unfolded and rendered to fragments; as this program proceeds, the substrate passes from site to specialized site within the complex. The 20S proteasome has been characterized in great detail, whereas relatively little is known about the 19S regulator. Structural analysis of the complete 26S proteasome is hampered by its instability, which makes it notoriously difficult
Occurrence and subunit composition of 20S proteasomes
As the number of sequenced genomes grows, a progressively clearer picture of the species distribution of proteasomes is emerging. In general, it appears that proteasomes are ubiquitous and essential in eukaryotes; ubiquitous, but not essential in archaea [2]; and rare and nonessential in bacteria [3], in which other energy-dependent proteases abound (to date, genuine bacterial proteasomes have been identified only in actinomycetales [4]). Typical archaeal proteasomes, for example, from Thermoplasma acidophilum, are built from single species of α and β subunits, although, more recently, duplicated genes encoding additional α- or β-type subunits have been identified in some cases [5]. Similarly, proteasomes from actinomycetales, for example, Mycobacterium, Streptomyces and Frankia, are composed of single α and β subunits [3,6,7], except for Rhodococcus erythropolis, which has two subunits of each kind [8]. Yeast proteasomes are composed of seven distinct α-type subunits and seven distinct β-type subunits. In higher eukaryotes, γ-interferon induces the exchange of three β-type subunits for three related β-type subunits [9]. Despite these differences in complexity, the architecture of 20S proteasomes is highly conserved [10••]. Structure and function of 20S proteasomes
The 20S proteasome is barrel-shaped, with a diameter of 11 nm, a height of 15 nm and a mass of 700 kDa. The barrel is a bipolar stack of four seven-membered rings, the two outer rings being formed by α subunits and the two inner rings by β subunits. As most prokaryotic proteasomes have single species of both subunits, they form homomeric rings, whereas eukaryotic proteasomes contain heteromeric rings formed by their seven distinct α- and β-type subunits [10••]. The central β rings enclose a cavity, approximately 5 nm in diameter, that houses the active sites: this inner compartment is a key structural feature common to all self-compartmentalizing proteases so far characterized (for a recent review, see [11]).
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In prokaryotic proteasomes, the proteolytic active site includes the N-terminal threonine of the β subunit [12], which is exposed after autocatalytic removal of its propeptide [13]. Only three of the seven eukaryotic β subunits, namely β1, β2 and β5, have an N-terminal threonine, resulting in only six active sites per proteasome [14]. A mutational and crystallographic study of yeast proteasomes found that the inactive β-type subunits, β3, β6 and β7, cannot be rendered active by introducing canonically active residues, including Thr1 [15•]. Thus, additional changes are necessary to allow the inactive subunits to adopt a fold that is conducive to proteolysis. Mutational studies and analyses with peptide and protein substrates demonstrated that the three active subunits have different specificities, that is, chymotrypsin-like, trypsin-like and peptidyl-glutamyl peptide hydrolyzing activity [16•,17•]. Nevertheless, the nature of all the active site residues is canonical, as predicted [12]. More recently, a cyclical ‘bite–chew’ model for protein breakdown by the 20S proteasome was suggested, based on the discovery of mutual allosteric regulation of the chymotrypsin-like and peptidyl-glutamyl peptide hydrolyzing active sites [18•].
eukaryotes, acquired the 20S proteasome by horizontal gene transfer from a host organism [21]. The human α7 subunit forms a ring-like complex when expressed alone and a heteromeric complex when co-expressed with its neighbors, α6 and α1 [22], implying that it has an important role in assembly. The propeptides of the β subunits are processed during their assembly into 20S proteasomes [23,24]. The first characterized intermediate of 20S proteasome assembly was the 300 kDa or 13S complex, which consists of all the α subunits and a subset of β subunits (β2, β3 and β4) [25,26]. Incorporation of the remaining β subunits (β1, β5–β7) triggers dimerization and concomitant processing of the propeptides, yielding fully assembled active proteasomes [25,26]. In yeast, a protein called Ump1p acts as a chaperone in the processing of the β5 propeptide and is thereafter degraded by the proteasome [27•]. Mutational and structural analysis showed that another function of the propeptides, besides driving assembly of the 20S core, is to prevent the Nα-acetylation of the N-terminal catalytic threonine residues, which would result in inhibition of the active subunits [15•,24,28•].
Assembly pathway
Mode of degradation and the size of products
The assembly of 20S proteasomes from R. erythropolis has served as a model for bacterial proteasome assembly in general. Their two α-type and two β-type subunits assemble into active proteasomes when a single α and a single β subunit are co-expressed in any of the four possible combinations [8]. The two β subunits are translated as precursors with propeptides of 65 and 59 residues. Individually expressed, both the α and β subunits remain monomeric and β-type precursors remain unprocessed; however, when mixed, they assemble immediately. This suggests that the first intermediate is a heterodimer of an α subunit and a β-subunit precursor. To date, the earliest intermediate actually demonstrated is the half-proteasome, composed of one ring each of the α subunits and β-subunit precursors. Halfproteasomes remain inactive, even when the β propeptide is deleted, indicating that active sites form only after the dimerization of half-proteasomes.
Various substrate proteins are degraded in a processive manner by proteasomes from Thermoplasma, yeast and rabbit muscle [17•,29,30]. The generated products are between 3 and 30 amino acids in length, with an average of 7 to 8 residues, independent of the number, specificity or spatial arrangement of the active sites [17•,30,31].
The β propeptides are not essential for assembly, but their absence retards proteasome formation [19]. They can exert their function whether covalently attached at the N terminus or added as free peptides. The co-expression of α subunits and β-subunit precursors that are unable to cleave their propeptides yields preholo-proteasomes — fully assembled particles in which the propeptides fill the central cavities and the antechambers [20]. Preholo-proteasomes are considered to be the final intermediate; their docking triggers the autocatalytic processing of the propeptides and active site formation. The cleaved propeptides are degraded in a processive manner, like substrate proteins [20]. In many respects, the assembly pathway of Rhodococcus proteasomes is similar to that of eukaryotic proteasomes. This is in line with the hypothesis that actinomycetes, many of which are symbionts or parasites in close contact with
Structure and function of the 26S proteasome The 19S regulatory subcomplex prepares protein substrates for degradation by the 20S core. The regulator is involved in the recognition and binding of substrate proteins, deubiquitination, unfolding and translocation into the core subcomplex. Translocation is thought to be coupled to the gating of the axial entry channel into the proteolytic cavity. Three-dimensional reconstruction by electron microscopy
Some progress was made recently in elucidating the structural organization of the 26S proteasome. A threedimensional model of the 26S proteasome from Drosophila embryos was obtained using electron microscopy in conjunction with image reconstruction [32••]. This analysis revealed a rather flexible linkage between the 19S regulator and the 20S core, described as a ‘wagging-type’ relative movement, that is influenced by the presence or absence of Mg–ATP. The images analyzed were of particles immobilized by adsorption to a carbon film and it may be that more complex movements occur under physiological conditions. The density map of the 19S regulator revealed a porous structure that does not immediately suggest a path for a protein substrate to follow en route to the core. The 19S regulator consists of lid and base subcomplexes
Biochemical and genetic analysis, in combination with electron microscopy and image analysis, of yeast 26S
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proteasomes led to the structural dissection of the 19S regulator into two subcomplexes, the ‘lid’ and the ‘base’ [33••] (Figure 1). Deletion of the 19S regulator subunit Rpn10 yields 26S proteasomes from which a complex of eight subunits, called the lid, can be dissociated at high salt concentrations. Independently, the lid subcomplex was isolated as a free particle from human erythrocytes and was shown to have the same subunit composition [34]. Interestingly, the eight subunits of the lid share significant sequence similarity with the eight subunits of the COP9 signalosome, a regulatory complex with kinase activity in mammals and plants that has been implicated in signal transduction [34–37]. Thus, the lid and the signalosome appear to have evolved from a common ancestor into molecules with distinct functions. The second subcomplex, the base, remains associated with the 20S core under the above-mentioned conditions: it is built from six functionally nonredundant ATPases (Rpt1–6) [38•] and the two largest subunits (Rpn1 and Rpn2) of the 26S proteasome [33••]. The proteasome–base complex is deficient in the degradation of ubiquitinated
substrates, but is still effective in the ATP-dependent degradation of nonubiquitinated substrates. Thus, the lid links the machinery for energy-dependent protein degradation — the core–base complex — to the ubiquitin system, which confers specificity. Chaperone-like activity of the base
In a related study, it was demonstrated that the base of the 19S regulator has a chaperone-like activity, that is, it could bind and refold denatured citrate synthase in an ATPdependent reaction [39••]. This was a surprising finding, because the function of proteasomal ATPases was thought to be the opposite, namely unfolding of substrate proteins for degradation by the core particle [40]. Possibly, its lack of ubiquitin allowed the citrate synthase substrate to dissociate from the base–proteasome complex and refold, thus escaping the degradation machine. In addition, the Janus-faced base [40] might work in both unfoldase and foldase modes, with the fate of the substrate depending on whether or not the base is complexed with the 20S core. Remarkably, the enzyme ornithine decarboxylase (ODC) is degraded by the 26S proteasome in an ATP-dependent,
Figure 1 A comparison of the structures of the 26S proteasome and the ClpAP protease. (a) Model of the 26S proteasome obtained after combining the 3D reconstruction of the 19S regulator (blue) — the lid (distal) and base (proximal) subcomplexes are indicated by different shades of blue — from Drosophila [32••] with the crystal structure of the 20S core (yellow) from Thermoplasma [65]. (b) A model of the ClpAP protease from E. coli derived from the combination of the 3D reconstruction of the ClpAP protease (blue) [71••] with the crystal structure of the ClpP protease (yellow) [64]. The scale bar represents 10 nm.
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but ubiquitin-independent manner, although the protein cofactor antizyme is essential for the recognition of ODC by the 26S proteasome [41]. Energy-dependent unfolding and translocation of ODC into the 20S core is performed in a chaperone-like manner [42]. The modulator
A 220 kDa complex, called the modulator, consists of two proteasomal ATPases (Rpt4 and Rpt5) and the p27 protein, a subunit of the 26S proteasome [43,44]. This modulator enhances the proteolytic activity of proteasomes, most likely by stimulating the formation of doubly capped 26S particles [45]. However, the extra mass of the modulator (220 kDa) was not detected in averaged electron micrographs of supposed proteasome–modulator complexes. As mass of this order should be readily detectable by this technique, it remains unclear whether the modulator forms a stable complex with the 20S proteasome or whether it engages only transiently to facilitate the association of 19S regulators with 20S cores.
Prokaryotic proteasome regulators The 20S proteasome from Thermoplasma was found to degrade only short peptides and unfolded proteins [46]. In the search for the postulated regulatory complex, a 600 kDa ATPase from Methanococcus jannaschii, called PAN (proteasome activating nucleotidase), was identified that is closely related to the eukaryotic proteasomal ATPases [47•]. PAN activates the degradation of protein, but not of peptide, substrates by 20S proteasomes from several archaebacteria and eukaryotes. Image analysis of electron micrographs demonstrated that PAN is sixfold symmetric (P Zwickl et al., unpublished data). One hallmark of proteasomal ATPases is a predicted coiled-coil region at their N termini. Its function remains unclear, but suggestions include involvement in oligomerization, in the interaction between the regulator and the 20S core, and in substrate binding and translocation. For some related AAA ATPases (ATPases associated with a variety of cellular activities), it has been demonstrated that their N-terminal domains, although adopting distinct folds, do indeed interact with substrates [48••,49••,50,51]. It is puzzling that the occurrence of PAN is not strictly correlated with that of proteasomes. The genomes of T. acidophilum (A Ruepp, personal communication) and Pyrobaculum aerophilum (S Fitz-Gibbon, personal communication) contain the proteasomal α and β genes, but not a PAN homolog. As it is unlikely that these proteasomes can function without the assistance of energy-dependent regulators, there must be some functional homologs; prime candidates in Thermoplasma are VAT (valosine-containing protein-like ATPase from T. acidophilum) and LonH (a Lon homolog), both AAA proteins. LonH shares sequence similarity with the ATPase domain of the Lon protease, but lacks its proteolytic domain. VAT is related to the eukaryotic CDC48/p97 proteins, which are involved in homotypic membrane fusion.
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Recently, some progress has been made in characterizing VAT from Thermoplasma. Depending on the Mg ion concentration, VAT can act in either an unfolding or a refolding mode [52]. Structurally, VAT is remarkably similar to NSF (N-ethylmaleimide sensitive factor), another eukaryotic protein involved in membrane fusion. In both VAT and NSF, the two AAA domains (D1 and D2) form hexameric toroids [53,54]. The oligomerization (or D2) domain of NSF consists of a nucleotide-binding subdomain with a Rossmann fold and a helical C-terminal subdomain [55••,56••]. Unlike the similar AAA domains of VAT and NSF, their approximately 20 kDa N-terminal substrate-binding domains are rather divergent; nevertheless, they adopt very similar topologies [48••,49••,57••]. The solution structure of the VAT N-terminal domain reveals two half-domains, with the N-terminal subdomain forming a double Ψ barrel and the C-terminal subdomain forming a β clamp [57••]. In actinomycetes, a gene encoding an AAA ATPase that is distantly related to proteasomal ATPases was identified in the vicinity of the operon for the proteasome subunit genes [4]. The Rhodococcus ATPase forms 600 kDa rings with sixfold symmetry and was consequently dubbed ARC (ATPase forming ring-shaped complexes) [58]. The presence of an archetypal N-terminal coiled coil in ARC is a further indication of a functional interaction with proteasomes.
Bacterial ATP-dependent proteases In bacteria, energy-dependent proteolysis is carried out by multiple systems [1,4,59]. These cells generally contain caseinolytic protease (Clp) and Lon, as well as HslVU/ClpQY proteases, built from a protease (HslV/ClpQ) and an ATPase (HslU/ClpY) component, and, in a few cases, genuine 20S proteasomes. Clp proteases differ from proteasomes in terms of subunit composition and the fold of the protease subunits. Nevertheless, certain features of their architecture and functional organization are compellingly similar (and extend to HslVU/ClpQY) — most notably, the co-axial stacking of hexameric rings of ATPase subunits on one or both faces of the core protease (Figure 1). Assuming that this resemblance extends to the mode of action (i.e. the ‘disassembly and degradation’ line), Clp proteases serve — at least, for some phenomena — as scaled down and simplified models of the proteasome. Lon, on the other hand, has the basic difference that its protease and ATPase domains are connected on the same polypeptide chain. It assembles into a flexible oligomer, reported to be a heptamer in yeast [60•]. Comparative properties of Clp proteases and proteasomes
The core of the modular ClpAP and ClpXP proteases from Escherichia coli is a double heptamer of ClpP subunits, which can associate with the hexameric ClpA or ClpX ATPase complex [61,62]. Sevenfold symmetry was also demonstrated recently for human mitochondrial ClpP [63]. The hollow core of this complex houses its proteolytic
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sites. The crystal structure of ClpP [64] revealed a subunit fold quite distinct from that of the 20S proteasome [14,65]. In essence, ClpP may be considered to be an analog of the 20S proteasome, but with a different subunit fold and without the outer rings of α subunits. Although its active site, as a serine protease, also differs from that of the proteasome, a threonine protease, it nevertheless contrives to generate peptides of a similar length to the 20S proteasome in processive degradation [66]. Subunit composition
The range of organisms in which homologs of Clp protease subunits have been identified continues to grow and now includes two bacterial viruses [67,68]. Some cases of multiple genes for ClpP have been documented, for example, in Streptomyces coelicolor [69] and Arabidopsis [70]. It will be of interest to learn whether these subunits segregate into homomeric rings, as in E. coli, or whether they combine in heteromeric rings, as in the Rhodococcus 20S proteasome. The ATPases ClpA and ClpX are homohexamers that require nucleotide to be stabilized in the assembled form. The ClpA subunit has two ATPase domains, which, in the hexamer, form two tiers with a cavity between them [71••]. ClpX has a single ATPase domain and forms a single-tiered ring [72]. Specificity
Self-compartmentalization is the principal mechanism whereby proteins that are not specifically targeted for degradation by ClpP or HslV/ClpQ are shielded from their active sites. As with the 20S proteasome, the proteolytic compartment of ClpP is accessible only through a channel that is so narrow (~10 Å) [64] that it may be negotiated only by unfolded polypeptides. Consistent with this scenario, ClpP on its own is capable of degrading peptides with up to about 30 amino acids. The degradation of larger folded protein substrates requires the collusion of an ATPase.
of NSF [55••,56••]. In the D2 hexamer, ATP is bound at the intersubunit interfaces. The absence of cognate sequences in expressed SSD domains of Clp proteases would explain their failure to oligomerize [74••]. In NSF, the binding of ATP by D2 results in oligomerization, whereas D1 — its other ATPase domain — hydrolyses ATP. A similar division of responsibilities has been attributed to the two ATPase domains of ClpA, but in reverse order [78]. In the same vein, the substrate-binding domain of NSF [48••,49••] is at the N terminus, in contrast with the C-terminal disposition of the SSD in ClpA. In the absence of protease, but the presence of nucleotide, the ATPases assemble to their hexameric state and can exercise unfolding or chaperonelike activity [79,80••]. As noted above, fully assembled and active complexes have ATPase rings bound on one or both faces of the protease. Thus positioned, they are strategically placed both to control which proteins have access to the channel leading into the proteolytic chamber and to supervise substrate translocation. In the processing of RepA, the substrate may bind either to ClpA [77••] or to preassembled ClpAP complexes, which can remain associated through multiple cycles of degradation [78]. The orientation of the ATPase rings relative to their proteases is yet to be established. In the case of ClpA, it has been suggested that its C-terminal domain binds to ClpP, on the grounds that this domain is most closely related to ClpX, which also binds ClpP [61]. In this orientation, the distal N-terminal domain would be exposed for substrate binding [77••]. On the other hand, the docking of the C-terminal domains on to ClpP may pose steric problems for ssrAdirected substrate binding by SSD domains, unless they are distributed around, rather than on the face of, the distal ring. Symmetry mismatch
Substrate selection
The presence of multiple energy-dependent proteases in E. coli has been seen as a means of extending the range of substrate specificity, but the system also accommodates some redundancy, as ClpAP, ClpXP and Lon have substrates in common [59,73]. An elegant mechanism has been discovered whereby an 11-residue tag called the ssrA (chromosomal 10Sa gene) becomes appended to the C termini of prematurely terminated proteins. This tag is recognized as a degradation signal by ClpA, ClpX and Lon via homologous sequences in their respective C termini — their so-called SSD (sensor and substrate discrimination) domains [74••]. ssrA tagging does not, however, cover the full range of substrate recognition mechanisms employed by Clp proteases. For certain phage proteins, for example, the λ O protein and the P1 RepA protein, as well as the bacterial SOS response protein UmuD, N-terminal binding has also been documented [75,76,77••]. States of assembly
The closest relative of a Clp ATPase for which a high-resolution crystal structure has been obtained is the D2 domain
An intriguing feature of the ClpAP complex is the discordant symmetries of the rings of the ClpP (sevenfold) and ClpA (sixfold) subunits. As these molecules stack axially, there must be a nonequivalent interaction between the respective rings. A similar mismatch apparently occurs in the proteasome at the interface between the 20S core (definitely sevenfold [14]) and the 19S regulator (putatively sixfold) (see Table 1). Comparable mismatches have been observed in a few other systems — notably, the DNA packaging vertex of bacteriophage capsids and the F1F0 proton transporter. During packaging, the DNA is translocated into the capsid by the terminase (another ATPase) via a connector/portal oligomer embedded in the capsid shell. To date, all connectors that have been convincingly demonstrated to be assembly competent are 12-fold rings that, in their respective capsids, occupy sites of fivefold symmetry. The φ29 connector — the most comprehensively studied such molecule — has been confirmed as a 12-fold ring by high-resolution atomic force microscopy [81] and crystallographic rotation function
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Table 1 Symmetry in subcomplexes of ATP-dependent proteases. Protease subcomplex Eukaryotic 26S proteasome Pseudo 7 Archaeal proteasome–PAN 7 Bacterial proteasome–ARC† 7 Bacterial ClpP–ClpA or –ClpX 7 Human mitochondrial ClpP 7 Bacterial HslV/ClpQ–HslU/ClpY 6 Yeast mitochondrial Lon 7§
Regulator subcomplex Most likely pseudo 6* 6 6 6 ND 6 and 7‡ 7§
*Six proteasomal ATPase genes are found in the yeast genome. † An interaction between ARC and the proteasome has not been demonstrated yet. ‡ Overexpressed HslU forms sixfold and sevenfold symmetric particles. §Protease and ATPase domains are linked on a single polypeptide chain. ND, not determined.
analysis [82]. Alternatively, in one case (bacteriophage SPP1), overexpressed connectors have been reported to be 13-fold rings [83] and, in another, bacteriophage T7, to be a mixture of 12-fold and 13-fold rings [84]. It is not yet clear whether the 13-mers are assembly competent: if not, their order of symmetry most likely represents an artifactual consequence of overexpression. Another such mismatch occurs within F1F0 [85], in which the pseudo-threefold F1-ATPase overlies the intramembranous F0 component, which has a much higher order of symmetry (10- or 12-fold) [86,87]. In both systems, the mismatch has been implicated in relative rotational movement [88,89] and such rotation has been elegantly demonstrated in the F1F0 system [90,91]. Returning to energy-dependent proteases, it is appealing to equate the symmetry mismatch with the relative rotation of the ATPase and the protease during processive Figure 2 Axial view of the ClpAP complex at 29 Å resolution [71••]. ClpP (yellow) has sevenfold symmetry and ClpA (blue) has sixfold symmetry, so that the interactions between pairs of subunits in the two rings are nonequivalent. Their binding is inferred to be dependent on a key interaction (white asterisk) between one specific subunit in ClpA (red dot) and one ClpP subunit (green dot). Relative rotation by only 8.6° (equivalent to a shift of 4.5 Å at the point of contact) transfers the key relationship to neighboring subunits. Thus, the symmetry mismatch is conducive to relative rotation without disengagement of the two subcomplexes. Such rotation might occur during the processive translocation of substrates into ClpP [71••]. A similar symmetry mismatch occurs in the 26S proteasome and some other energy-dependent proteases (see Table 1).
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digestion of substrates. In the ClpAP system, the relative angular disposition of the ClpP and ClpA rings has been determined [71••]. This interaction is based on key subunits in both rings. Key status is transferred to neighboring subunits by a small rotation (8°), corresponding to a translational shift of only 4–5 Å at the point of contact (Figure 2), a property that facilitates rotation without separation of the subcomplexes. On the other hand, HslVU/ClpQY complexes assembled in vitro exhibit 6:6, as well as 6:7, symmetry combinations ([92,93]; see Table 1). Lon, whose ATPase and protease domains are covalently linked, inevitably requires equimolarity in active complexes. For questions pertaining to the functional implications of symmetry mismatches for processive, energy-dependent proteolysis, final answers are unlikely to precede further experiments.
Conclusions Common to all known energy-dependent proteases is a linear arrangement of the proteolytic modules and the regulatory modules, which prepare substrates for degradation. These tasks include substrate recognition, unfolding and translocation. Although the exact role of ATP hydrolysis remains to be established, it is likely that the energy-dependent steps are unfolding and translocation. Although not universal, a recurring feature is a symmetry mismatch between the ATPase module and the proteolytic module. This property, which occurs in the proteasome, as well as in the ClpAP and ClpXP proteases, has led to the hypothesis that the symmetry mismatch may facilitate relative rotation of the respective modules in the processive degradation of substrates. However, there is no such mismatch in Lon nor, putatively, in ClpYQ (HslUV) [94••]. This may indicate that processivity in the latter complexes is effected by movements involving axial translations, rather than relative motions between the respective modules.
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Acknowledgements We thank Sorel Fitz-Gibbon and Andreas Ruepp for communication of unpublished results and David Belnap and Kornelius Zeth for help with the graphics.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
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Nagy I, Tamura T, Vanderleyden J, Baumeister W, De Mot R: The 20S proteasome of Streptomyces coelicolor. J Bacteriol 1998, 180:5448-5453.
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17. •
Nussbaum AK, Dick TP, Keilholz W, Schirle M, Stevanovic S, Dietz K, Heinemeyer W, Groll M, Wolf DH, Huber R et al.: Cleavage motifs of the yeast 20S proteasome β subunits deduced from digests of enolase 1. Proc Natl Acad Sci USA 1998, 95:12504-12509. The authors describe the analysis of the cleavage products of enolase degraded by yeast proteasomes. A comparison of the fragments generated by wild-type and mutant proteasomes demonstrated that the product length is not dependent on the number of active sites. 18. Kisselev AF, Akopian TN, Castillo V, Goldberg AL: Proteasome active • sites allosterically regulate each other, suggesting a cyclical bitechew mechanism for protein breakdown. Mol Cell 1999, 4:395-402. A cyclical bite–chew mechanism for protein breakdown by proteasomes is proposed, based on the finding of allosteric regulation of active sites. 19. Zühl F, Seemüller E, Golbik R, Baumeister W: Dissecting the assembly pathway of the 20S proteasome. FEBS Lett 1997, 418:189-194. 20. Mayr J, Seemüller E, Müller SA, Engel A, Baumeister W: Late events in the assembly of 20S proteasomes. J Struct Biol 1998, 124:179-188. 21. Lupas A, Zühl F, Tamura T, Wolf S, Nagy I, De Mot R, Baumeister W: Eubacterial proteasomes. Mol Biol Rep 1997, 24:125-131. 22. Gerards WLH, Dejong WW, Boelens W, Bloemendal H: Structure and assembly of the 20S proteasome. Cell Mol Life Sci 1998, 54:253-262. 23. Schmidt M, Zantopf D, Kraft R, Kostka S, Preissner R, Kloetzel PM: Sequence information within proteasomal prosequences mediates efficient integration of β-subunits into the 20 S proteasome complex. J Mol Biol 1999, 288:117-128. 24. Jäger S, Groll M, Huber R, Wolf DH, Heinemeyer W: Proteasome β-type subunits: unequal roles of propeptides in core particle maturation and a hierarchy of active site function. J Mol Biol 1999, 291:997-1013. 25. Nandi D, Woodward E, Ginsburg DB, Monaco JJ: Intermediates in the formation of mouse 20S proteasomes - implications for the assembly of precursor β-subunits. EMBO J 1997, 16:5363-5375. 26. Schmidtke G, Schmidt M, Kloetzel PM: Maturation of mammalian 20 S proteasome: purification and characterization of 13 S and 16 S proteasome precursor complexes. J Mol Biol 1997, 268:95-106. 27. •
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and related to the COP9-signalosome and eIF3. Cell 1998, 94:615-623. The dissection of the 19S regulator of the yeast 26S proteasome into ‘lid’ and ‘base’ subcomplexes using functional and structural analyses. The lid is essential for the degradation of ubiquitinated substrates, whereas the base is responsible for ATP-dependent degradation. 34. Henke W, Ferrell K, Bech-Otschir D, Seeger M, Schade R, Jungblut P, Naumann M, Dubiel W: Comparison of human COP9 signalosome and 26S proteasome ‘lid’. Mol Biol Rep 1999, 26:29-34. 35. Seeger M, Kraft R, Ferrell K, Bech-Otschir D, Dumdey R, Schade R, Gordon C, Naumann M, Dubiel W: A novel protein complex involved in signal-transduction possessing similarities to 26S proteasome subunits. FASEB J 1998, 12:469-478.
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Zwickl P, Ng D, Woo KM, Klenk H-P, Goldberg AL: An archaebacterial ATPase, homologous to the ATPases in the eukaryotic 26S proteasome, activates protein breakdown by the 20S proteasome. J Biol Chem 1999, 274:26008-26014. The first characterization of an archaebacterial regulatory nucleotidase complex that stimulates protein breakdown by archaeal proteasomes. 48. May AP, Misura KMS, Whiteheart SW, Weis WI: Crystal structure of •• the amino-terminal domain of N-ethylmaleimide-sensitive fusion protein. Nat Cell Biol 1999, 1:175-182. The crystal structure of the N-terminal substrate-binding domain of the N-ethylmaleimide sensitive factor (NSF) was determined independently by May et al. [48••] and Yu et al. [49••]. The N-terminal half of the substratebinding domain folds into a double-ψ β barrel, whereas the C-terminal half adopts a four-strand–one-helix α/β roll. The two subdomains form a groove that is proposed to be the substrate (SNAP) interaction site. See also annotation to [57••].
55. Lenzen CU, Steinmann D, Whiteheart SW, Weis WI: Crystal •• structure of the hexamerization domain of N-ethylmaleimidesensitive fusion protein. Cell 1998, 94:525-536. The structure of the second nucleotide-binding domain of the AAA ATPase N-ethylmaleimide sensitive factor in the presence of nucleotide was determined in parallel by Lenzen et al. [55••] and Yu et al. [56••]. The fold of the protomers revealed a nucleotide-binding domain with a Rossmann fold and a helical domain. 56. Yu RC, Hanson PI, Jahn R, Brünger AT: Structure of the ATP •• dependent oligomerization domain of N-ethylmaleimide sensitive factor complexed with ATP. Nat Struct Biol 1998, 5:803-811. See annotation to [55••]. 57. ••
Coles M, Diercks T, Liermann J, Gröger A, Rockel B, Baumeister W, Koretke KK, Lupas A, Peters J, Kessler H: The solution structure of VAT-N reveals a ‘missing link’ in the evolution of complex enzymes from a simple element. Curr Biol 1999, 9:1158-1168. NMR spectroscopy revealed that the N-terminal substrate-recognition domain of VAT is composed of two subdomains. The N-terminal subdomain forms a double-ψ β barrel and the C-terminal subdomain adopts a novel sixstranded β-clam fold. A path for the evolution of the double-ψ β-barrel fold is proposed. See also annotations to [48••,49••]. 58. Wolf S, Nagy I, Lupas A, Pfeifer G, Cejka Z, Müller SA, Engel A, De Mot R, Baumeister W: Characterization of ARC, a divergent member of the AAA ATPase family from Rhodococcus erythropolis. J Mol Biol 1998, 277:13-25. 59. Gottesman S, Wickner S, Maurizi MR: Protein quality control: triage by chaperones and proteases. Genes Dev 1997, 11:815-823. 60. Stahlberg H, Kutejova E, Suda K, Wolpensinger B, Lustig A, Schatz G, • Engel A, Suzuki CK: Mitochondrial Lon of Saccharomyces cerevisiae is a ring-shaped protease with seven flexible subunits. Proc Natl Acad Sci USA 1999, 96:6787-6790. This structural study of Lon protease from yeast by electron microscopy and analytical ultracentrifugation supports the view that this enzyme, with covalently linked protease and ATPase domains, forms a flexible heptameric ring. 61. Kessel M, Maurizi MR, Kim B, Kocsis E, Trus BL, Singh SK, Steven AC: Homology in structural organization between E. coli ClpAP protease and the eukaryotic 26 S proteasome. J Mol Biol 1995, 250:587-594. 62. Flanagan JM, Wall JS, Capel MS, Schneider DK, Shanklin J: Scanning transmission electron microscopy and small-angle scattering provide evidence that native Escherichia coli ClpP is a tetradecamer with an axial pore. Biochemistry 1995, 34:10910-10917. 63. de Sagarra MR, Mayo I, Marco S, Rodriguez-Vilarino S, Oliva J, Carrascosa JL, Castano JG: Mitochondrial localization and oligomeric structure of HClpP, the human homologue of E. coli ClpP. J Mol Biol 1999, 292:819-825. 64. Wang J, Hartling JA, Flanagan JM: The structure of ClpP at 2.3 Å resolution suggests a model for ATP-dependent proteolysis. Cell 1997, 91:447-456. 65. Löwe J, Stock D, Jap B, Zwickl P, Baumeister W, Huber R: Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 Å resolution. Science 1995, 268:533-539.
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Desiere F, Lucchini S, Brüssow H: Comparative sequence analysis of the DNA packaging, head, and tail morphogenesis modules in the temperate cos-site Streptococcus thermophilus bacteriophage Sfi21. Virology 1999, 260:244-253.
68. Gilakjan ZA, Kropinski AM: Cloning and analysis of the capsid morphogenesis genes of Pseudomonas aeruginosa bacteriophage D3: another example of protein chain mail? J Bacteriol 1999, 181:7221-7227.
78. Singh SK, Guo FS, Maurizi MR: ClpA and ClpP remain associated during multiple rounds of ATP dependent protein degradation by ClpAP protease. Biochemistry 1999, 38:14906-14915. 79. Jones JM, Welty DJ, Nakai H: Versatile action of Escherichia coli ClpXP as protease or molecular chaperone for bacteriophage Mu transposition. J Biol Chem 1998, 273:459-465. 80. Weber-Ban EU, Reid BG, Miranker AD, Horwich AL: Global •• unfolding of a substrate protein by the Hsp100 chaperone ClpA. Nature 1999, 401:90-93. The unfolding of substrates by ClpA in the absence of proteolysis was demonstrated in a fluorescence assay using ssrA-tagged green fluorescent protein as a substrate. The substrate was stabilized in the unfolded state by being subsequently bound to a modified chaperone.
69. de Crecy-Lagard V, Servant-Moisson P, Viala J, Grandvalet C, Mazodier P: Alteration of the synthesis of the Clp ATP-dependent protease affects morphological and physiological differentiation in Streptomyces. Mol Microbiol 1999, 32:505-517.
81. Müller DJ, Engel A, Carrascosa JL, Velez M: The bacteriophage φ29 head-tail connector imaged at high resolution with the atomic force microscope in buffer solution. EMBO J 1997, 16:2547-2553.
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82. Guasch A, Pous J, Parraga A, Valpuesta JM, Carrascosa JL, Coll M: Crystallographic analysis reveals the 12-fold symmetry of the bacteriophage φ29 connector particle. J Mol Biol 1998, 281:219-225. 83. Dube P, Tavares P, Lurz R, van Heel M: The portal protein of bacteriophage SPP1: a DNA pump with 13-fold symmetry. EMBO J 1993, 12:1303-1309.
71. Beuron F, Maurizi MR, Belnap DM, Kocsis E, Booy FP, Kessel M, •• Steven AC: At sixes and sevens: characterization of the symmetry mismatch of the ClpAP chaperone-assisted protease. J Struct Biol 1998, 124:179-188. This paper presents a cryo-electron microscopy reconstruction of ClpA, a two-domain ATPase, that reveals an internal compartment enclosed between two hexameric rings. It draws on the crystal structure of the double-heptamer protease ClpP [64] to determine their docking. A rotational model of processive substrate processing is suggested.
84. Kocsis E, Cerritelli ME, Trus BL, Cheng N, Steven AC: Improved methods for determination of rotational symmetries in macromolecules. Ultramicroscopy 1995, 60:219-228.
72. Grimaud R, Kessel M, Beuron F, Steven AC, Maurizi MR: Enzymatic and structural similarities between the Escherichia coli ATPdependent proteases, ClpXP and ClpAP. J Biol Chem 1998, 273:12476-12481.
86. Rastogi VK, Girvin ME: Structural changes linked to proton translocation by subunit c of the ATP synthase. Nature 1999, 402:263-268.
73. Hoskins JR, Pak M, Maurizi MR, Wickner S: The role of the ClpA chaperone in proteolysis by ClpAP. Proc Natl Acad Sci USA 1998, 95:12135-12140. 74. Smith CK, Baker TA, Sauer RT: Lon and Clp family proteases and •• chaperones share homologous substrate-recognition domains. Proc Natl Acad Sci USA 1999, 96:6678-6682. The homologous SSD (sensor and substrate discrimination) sequences of five ATP-dependent bacterial proteases were expressed in E. coli. Three of them were demonstrated to assume compact folds, providing support for the hypothesis that SSD sequences correspond to discrete domains. The binding of ssrA-tagged substrates by these domains was found to be partially selective. 75. Zylicz M, Liberek K, Wawrzynow A, Georgopoulos C: Formation of the preprimosome protects λ O from RNA transcriptiondependent proteolysis by ClpP/ClpX. Proc Natl Acad Sci USA 1998, 95:15259-15263. 76. Gonciarz-Swiatek M, Wawrzynow A, Um SJ, Learn BA, McMacken R, Kelley WL, Georgopoulos C, Sliekers O, Zylicz M: Recognition, targeting, and hydrolysis of the λ O replication protein by the ClpP/ClpX protease. J Biol Chem 1999, 274:13999-14005. 77. ••
Pak M, Hoskins JR, Singh SK, Maurizi MR, Wickner S: Concurrent chaperone and protease activities of ClpAP and the requirement for the N-terminal ClpA ATP binding site for chaperone activity. J Biol Chem 1999, 274:19316-19322. The authors exploited specific point mutations in the ATPase ClpA to study its interaction with the model substrate RepA, distinguishing the roles of its two ATPase domains in substrate binding and the dual roles of ClpA in either unfolding alone or unfolding as an activity in protein degradation.
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88. Boyer PD: A model for conformational coupling of membrane potential and proton translocation to ATP synthesis and to active transport. FEBS Lett 1975, 58:1-6. 89. Hendrix RW: Symmetry mismatch and DNA packaging in large bacteriophages. Proc Natl Acad Sci USA 1978, 75:4779-4783. 90. Noji H, Yasuda R, Yoshida M, Kinosita K Jr: Direct observation of the rotation of F1-ATPase. Nature 1997, 386:299-302. 91. Sambongi Y, Iko Y, Tanabe M, Omote H, Iwamoto-Kihara A, Ueda I, Yanagida T, Wada Y, Futai M: Mechanical rotation of the c subunit oligomer in ATP synthase (F0F1): direct observation. Science 1999, 286:1722-1724. 92. Rohrwild M, Pfeifer G, Santarius U, Müller SA, Huang HC, Engel A, Baumeister W, Goldberg AL: The ATP-dependent HslVU protease from Escherichia coli is a four-ring structure resembling the proteasome. Nat Struct Biol 1997, 4:133-139. 93. Bochtler M, Ditzel L, Groll M, Huber R: Crystal structure of heat shock locus V (HslV) from Escherichia coli. Proc Natl Acad Sci USA 1997, 94:6070-6074. 94. Bochtler M, Hartmann C, Song HK, Bourenkov GP, Bartunik HD, •• Huber R: The structures of HslU and the ATP-dependent protease HslU-HslV. Nature 2000, 403:800-806. Crystal structures of the HslU ATPase in its free form and in complex with the HslV protease were determined. In both structures, the HslU ATPase has a sixfold symmetry, as does the HslV protease, suggesting that a symmetry mismatch is nonexistent in the HslUV protease.
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Dis-assembly lines: the proteasome and related ATPase-assisted proteases Peter Zwickl*, Wolfgang Baumeister*† and Alasdair Steven‡ Self-compartmentalizing proteases, such as the proteasome and several prokaryotic energy-dependent proteases, are designed to act in the crowded environment of the cell. Proteins destined for degradation are recognized and unfolded by regulatory subcomplexes that invariably contain ATPase modules, before being translocated into another subcomplex, the proteolytic core, for degradation. The sequential actions effected on substrates are reflected in the linear arrangement of these subcomplexes; thus, the holocomplexes are organized as molecular disassembly and degradation lines.
to obtain homogeneous preparations. This lability is attributable primarily to the 19S component, whose complexity — it is built of at least 17 distinct subunits — poses a further challenge, exacerbated by its lack of symmetry. In this review, we summarize some recent advances that deepen our understanding of the structure, assembly and function of the 20S proteasome, but the main emphasis is on properties of the 19S particle. We also develop a comparison between proteasomes and simpler, but functionally related, complexes of bacterial descent.
Addresses *Department of Molecular Structural Biology, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany † e-mail: [email protected] ‡ Laboratory of Structural Biology, Building 6, Room B2-34, Center Drive, MSC-2717, NIAMS, National Institutes of Health, Bethesda, MD 20892-2717, USA
20S proteasomes
Current Opinion in Structural Biology 2000, 10:242–250 0959-440X/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations AAA ATPases associated with a variety of cellular activities ARC ATPase forming ring-shaped complexes Clp caseinolytic protease NSF N-ethylmaleimide sensitive factor ODC ornithine decarboxylase PAN proteasome activating nucleotidase SSD sensor and substrate discrimination ssrA chromosomal 10Sa gene VAT valosine-containing protein-like ATPase from Thermoplasma acidophilum
Introduction In eukaryotes, the 26S proteasome is the central protease in the energy-dependent degradation of proteins via the ubiquitin pathway. This quintessential molecular machine, with a cumulative mass of more than 2.5 MDa, comprises two subcomplexes — the 20S core particle and the 19S regulatory particle. The core particle performs the actual proteolysis, whereas the 19S particle recognizes and unfolds substrates and delivers them for degradation [1]. These activities are coordinated in a manner reminiscent of an industrial assembly line acting in reverse: in this case, the starting material — a protein targeted for degradation — is a complex entity that is sequentially dismantled, unfolded and rendered to fragments; as this program proceeds, the substrate passes from site to specialized site within the complex. The 20S proteasome has been characterized in great detail, whereas relatively little is known about the 19S regulator. Structural analysis of the complete 26S proteasome is hampered by its instability, which makes it notoriously difficult
Occurrence and subunit composition of 20S proteasomes
As the number of sequenced genomes grows, a progressively clearer picture of the species distribution of proteasomes is emerging. In general, it appears that proteasomes are ubiquitous and essential in eukaryotes; ubiquitous, but not essential in archaea [2]; and rare and nonessential in bacteria [3], in which other energy-dependent proteases abound (to date, genuine bacterial proteasomes have been identified only in actinomycetales [4]). Typical archaeal proteasomes, for example, from Thermoplasma acidophilum, are built from single species of α and β subunits, although, more recently, duplicated genes encoding additional α- or β-type subunits have been identified in some cases [5]. Similarly, proteasomes from actinomycetales, for example, Mycobacterium, Streptomyces and Frankia, are composed of single α and β subunits [3,6,7], except for Rhodococcus erythropolis, which has two subunits of each kind [8]. Yeast proteasomes are composed of seven distinct α-type subunits and seven distinct β-type subunits. In higher eukaryotes, γ-interferon induces the exchange of three β-type subunits for three related β-type subunits [9]. Despite these differences in complexity, the architecture of 20S proteasomes is highly conserved [10••]. Structure and function of 20S proteasomes
The 20S proteasome is barrel-shaped, with a diameter of 11 nm, a height of 15 nm and a mass of 700 kDa. The barrel is a bipolar stack of four seven-membered rings, the two outer rings being formed by α subunits and the two inner rings by β subunits. As most prokaryotic proteasomes have single species of both subunits, they form homomeric rings, whereas eukaryotic proteasomes contain heteromeric rings formed by their seven distinct α- and β-type subunits [10••]. The central β rings enclose a cavity, approximately 5 nm in diameter, that houses the active sites: this inner compartment is a key structural feature common to all self-compartmentalizing proteases so far characterized (for a recent review, see [11]).
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In prokaryotic proteasomes, the proteolytic active site includes the N-terminal threonine of the β subunit [12], which is exposed after autocatalytic removal of its propeptide [13]. Only three of the seven eukaryotic β subunits, namely β1, β2 and β5, have an N-terminal threonine, resulting in only six active sites per proteasome [14]. A mutational and crystallographic study of yeast proteasomes found that the inactive β-type subunits, β3, β6 and β7, cannot be rendered active by introducing canonically active residues, including Thr1 [15•]. Thus, additional changes are necessary to allow the inactive subunits to adopt a fold that is conducive to proteolysis. Mutational studies and analyses with peptide and protein substrates demonstrated that the three active subunits have different specificities, that is, chymotrypsin-like, trypsin-like and peptidyl-glutamyl peptide hydrolyzing activity [16•,17•]. Nevertheless, the nature of all the active site residues is canonical, as predicted [12]. More recently, a cyclical ‘bite–chew’ model for protein breakdown by the 20S proteasome was suggested, based on the discovery of mutual allosteric regulation of the chymotrypsin-like and peptidyl-glutamyl peptide hydrolyzing active sites [18•].
eukaryotes, acquired the 20S proteasome by horizontal gene transfer from a host organism [21]. The human α7 subunit forms a ring-like complex when expressed alone and a heteromeric complex when co-expressed with its neighbors, α6 and α1 [22], implying that it has an important role in assembly. The propeptides of the β subunits are processed during their assembly into 20S proteasomes [23,24]. The first characterized intermediate of 20S proteasome assembly was the 300 kDa or 13S complex, which consists of all the α subunits and a subset of β subunits (β2, β3 and β4) [25,26]. Incorporation of the remaining β subunits (β1, β5–β7) triggers dimerization and concomitant processing of the propeptides, yielding fully assembled active proteasomes [25,26]. In yeast, a protein called Ump1p acts as a chaperone in the processing of the β5 propeptide and is thereafter degraded by the proteasome [27•]. Mutational and structural analysis showed that another function of the propeptides, besides driving assembly of the 20S core, is to prevent the Nα-acetylation of the N-terminal catalytic threonine residues, which would result in inhibition of the active subunits [15•,24,28•].
Assembly pathway
Mode of degradation and the size of products
The assembly of 20S proteasomes from R. erythropolis has served as a model for bacterial proteasome assembly in general. Their two α-type and two β-type subunits assemble into active proteasomes when a single α and a single β subunit are co-expressed in any of the four possible combinations [8]. The two β subunits are translated as precursors with propeptides of 65 and 59 residues. Individually expressed, both the α and β subunits remain monomeric and β-type precursors remain unprocessed; however, when mixed, they assemble immediately. This suggests that the first intermediate is a heterodimer of an α subunit and a β-subunit precursor. To date, the earliest intermediate actually demonstrated is the half-proteasome, composed of one ring each of the α subunits and β-subunit precursors. Halfproteasomes remain inactive, even when the β propeptide is deleted, indicating that active sites form only after the dimerization of half-proteasomes.
Various substrate proteins are degraded in a processive manner by proteasomes from Thermoplasma, yeast and rabbit muscle [17•,29,30]. The generated products are between 3 and 30 amino acids in length, with an average of 7 to 8 residues, independent of the number, specificity or spatial arrangement of the active sites [17•,30,31].
The β propeptides are not essential for assembly, but their absence retards proteasome formation [19]. They can exert their function whether covalently attached at the N terminus or added as free peptides. The co-expression of α subunits and β-subunit precursors that are unable to cleave their propeptides yields preholo-proteasomes — fully assembled particles in which the propeptides fill the central cavities and the antechambers [20]. Preholo-proteasomes are considered to be the final intermediate; their docking triggers the autocatalytic processing of the propeptides and active site formation. The cleaved propeptides are degraded in a processive manner, like substrate proteins [20]. In many respects, the assembly pathway of Rhodococcus proteasomes is similar to that of eukaryotic proteasomes. This is in line with the hypothesis that actinomycetes, many of which are symbionts or parasites in close contact with
Structure and function of the 26S proteasome The 19S regulatory subcomplex prepares protein substrates for degradation by the 20S core. The regulator is involved in the recognition and binding of substrate proteins, deubiquitination, unfolding and translocation into the core subcomplex. Translocation is thought to be coupled to the gating of the axial entry channel into the proteolytic cavity. Three-dimensional reconstruction by electron microscopy
Some progress was made recently in elucidating the structural organization of the 26S proteasome. A threedimensional model of the 26S proteasome from Drosophila embryos was obtained using electron microscopy in conjunction with image reconstruction [32••]. This analysis revealed a rather flexible linkage between the 19S regulator and the 20S core, described as a ‘wagging-type’ relative movement, that is influenced by the presence or absence of Mg–ATP. The images analyzed were of particles immobilized by adsorption to a carbon film and it may be that more complex movements occur under physiological conditions. The density map of the 19S regulator revealed a porous structure that does not immediately suggest a path for a protein substrate to follow en route to the core. The 19S regulator consists of lid and base subcomplexes
Biochemical and genetic analysis, in combination with electron microscopy and image analysis, of yeast 26S
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proteasomes led to the structural dissection of the 19S regulator into two subcomplexes, the ‘lid’ and the ‘base’ [33••] (Figure 1). Deletion of the 19S regulator subunit Rpn10 yields 26S proteasomes from which a complex of eight subunits, called the lid, can be dissociated at high salt concentrations. Independently, the lid subcomplex was isolated as a free particle from human erythrocytes and was shown to have the same subunit composition [34]. Interestingly, the eight subunits of the lid share significant sequence similarity with the eight subunits of the COP9 signalosome, a regulatory complex with kinase activity in mammals and plants that has been implicated in signal transduction [34–37]. Thus, the lid and the signalosome appear to have evolved from a common ancestor into molecules with distinct functions. The second subcomplex, the base, remains associated with the 20S core under the above-mentioned conditions: it is built from six functionally nonredundant ATPases (Rpt1–6) [38•] and the two largest subunits (Rpn1 and Rpn2) of the 26S proteasome [33••]. The proteasome–base complex is deficient in the degradation of ubiquitinated
substrates, but is still effective in the ATP-dependent degradation of nonubiquitinated substrates. Thus, the lid links the machinery for energy-dependent protein degradation — the core–base complex — to the ubiquitin system, which confers specificity. Chaperone-like activity of the base
In a related study, it was demonstrated that the base of the 19S regulator has a chaperone-like activity, that is, it could bind and refold denatured citrate synthase in an ATPdependent reaction [39••]. This was a surprising finding, because the function of proteasomal ATPases was thought to be the opposite, namely unfolding of substrate proteins for degradation by the core particle [40]. Possibly, its lack of ubiquitin allowed the citrate synthase substrate to dissociate from the base–proteasome complex and refold, thus escaping the degradation machine. In addition, the Janus-faced base [40] might work in both unfoldase and foldase modes, with the fate of the substrate depending on whether or not the base is complexed with the 20S core. Remarkably, the enzyme ornithine decarboxylase (ODC) is degraded by the 26S proteasome in an ATP-dependent,
Figure 1 A comparison of the structures of the 26S proteasome and the ClpAP protease. (a) Model of the 26S proteasome obtained after combining the 3D reconstruction of the 19S regulator (blue) — the lid (distal) and base (proximal) subcomplexes are indicated by different shades of blue — from Drosophila [32••] with the crystal structure of the 20S core (yellow) from Thermoplasma [65]. (b) A model of the ClpAP protease from E. coli derived from the combination of the 3D reconstruction of the ClpAP protease (blue) [71••] with the crystal structure of the ClpP protease (yellow) [64]. The scale bar represents 10 nm.
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but ubiquitin-independent manner, although the protein cofactor antizyme is essential for the recognition of ODC by the 26S proteasome [41]. Energy-dependent unfolding and translocation of ODC into the 20S core is performed in a chaperone-like manner [42]. The modulator
A 220 kDa complex, called the modulator, consists of two proteasomal ATPases (Rpt4 and Rpt5) and the p27 protein, a subunit of the 26S proteasome [43,44]. This modulator enhances the proteolytic activity of proteasomes, most likely by stimulating the formation of doubly capped 26S particles [45]. However, the extra mass of the modulator (220 kDa) was not detected in averaged electron micrographs of supposed proteasome–modulator complexes. As mass of this order should be readily detectable by this technique, it remains unclear whether the modulator forms a stable complex with the 20S proteasome or whether it engages only transiently to facilitate the association of 19S regulators with 20S cores.
Prokaryotic proteasome regulators The 20S proteasome from Thermoplasma was found to degrade only short peptides and unfolded proteins [46]. In the search for the postulated regulatory complex, a 600 kDa ATPase from Methanococcus jannaschii, called PAN (proteasome activating nucleotidase), was identified that is closely related to the eukaryotic proteasomal ATPases [47•]. PAN activates the degradation of protein, but not of peptide, substrates by 20S proteasomes from several archaebacteria and eukaryotes. Image analysis of electron micrographs demonstrated that PAN is sixfold symmetric (P Zwickl et al., unpublished data). One hallmark of proteasomal ATPases is a predicted coiled-coil region at their N termini. Its function remains unclear, but suggestions include involvement in oligomerization, in the interaction between the regulator and the 20S core, and in substrate binding and translocation. For some related AAA ATPases (ATPases associated with a variety of cellular activities), it has been demonstrated that their N-terminal domains, although adopting distinct folds, do indeed interact with substrates [48••,49••,50,51]. It is puzzling that the occurrence of PAN is not strictly correlated with that of proteasomes. The genomes of T. acidophilum (A Ruepp, personal communication) and Pyrobaculum aerophilum (S Fitz-Gibbon, personal communication) contain the proteasomal α and β genes, but not a PAN homolog. As it is unlikely that these proteasomes can function without the assistance of energy-dependent regulators, there must be some functional homologs; prime candidates in Thermoplasma are VAT (valosine-containing protein-like ATPase from T. acidophilum) and LonH (a Lon homolog), both AAA proteins. LonH shares sequence similarity with the ATPase domain of the Lon protease, but lacks its proteolytic domain. VAT is related to the eukaryotic CDC48/p97 proteins, which are involved in homotypic membrane fusion.
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Recently, some progress has been made in characterizing VAT from Thermoplasma. Depending on the Mg ion concentration, VAT can act in either an unfolding or a refolding mode [52]. Structurally, VAT is remarkably similar to NSF (N-ethylmaleimide sensitive factor), another eukaryotic protein involved in membrane fusion. In both VAT and NSF, the two AAA domains (D1 and D2) form hexameric toroids [53,54]. The oligomerization (or D2) domain of NSF consists of a nucleotide-binding subdomain with a Rossmann fold and a helical C-terminal subdomain [55••,56••]. Unlike the similar AAA domains of VAT and NSF, their approximately 20 kDa N-terminal substrate-binding domains are rather divergent; nevertheless, they adopt very similar topologies [48••,49••,57••]. The solution structure of the VAT N-terminal domain reveals two half-domains, with the N-terminal subdomain forming a double Ψ barrel and the C-terminal subdomain forming a β clamp [57••]. In actinomycetes, a gene encoding an AAA ATPase that is distantly related to proteasomal ATPases was identified in the vicinity of the operon for the proteasome subunit genes [4]. The Rhodococcus ATPase forms 600 kDa rings with sixfold symmetry and was consequently dubbed ARC (ATPase forming ring-shaped complexes) [58]. The presence of an archetypal N-terminal coiled coil in ARC is a further indication of a functional interaction with proteasomes.
Bacterial ATP-dependent proteases In bacteria, energy-dependent proteolysis is carried out by multiple systems [1,4,59]. These cells generally contain caseinolytic protease (Clp) and Lon, as well as HslVU/ClpQY proteases, built from a protease (HslV/ClpQ) and an ATPase (HslU/ClpY) component, and, in a few cases, genuine 20S proteasomes. Clp proteases differ from proteasomes in terms of subunit composition and the fold of the protease subunits. Nevertheless, certain features of their architecture and functional organization are compellingly similar (and extend to HslVU/ClpQY) — most notably, the co-axial stacking of hexameric rings of ATPase subunits on one or both faces of the core protease (Figure 1). Assuming that this resemblance extends to the mode of action (i.e. the ‘disassembly and degradation’ line), Clp proteases serve — at least, for some phenomena — as scaled down and simplified models of the proteasome. Lon, on the other hand, has the basic difference that its protease and ATPase domains are connected on the same polypeptide chain. It assembles into a flexible oligomer, reported to be a heptamer in yeast [60•]. Comparative properties of Clp proteases and proteasomes
The core of the modular ClpAP and ClpXP proteases from Escherichia coli is a double heptamer of ClpP subunits, which can associate with the hexameric ClpA or ClpX ATPase complex [61,62]. Sevenfold symmetry was also demonstrated recently for human mitochondrial ClpP [63]. The hollow core of this complex houses its proteolytic
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sites. The crystal structure of ClpP [64] revealed a subunit fold quite distinct from that of the 20S proteasome [14,65]. In essence, ClpP may be considered to be an analog of the 20S proteasome, but with a different subunit fold and without the outer rings of α subunits. Although its active site, as a serine protease, also differs from that of the proteasome, a threonine protease, it nevertheless contrives to generate peptides of a similar length to the 20S proteasome in processive degradation [66]. Subunit composition
The range of organisms in which homologs of Clp protease subunits have been identified continues to grow and now includes two bacterial viruses [67,68]. Some cases of multiple genes for ClpP have been documented, for example, in Streptomyces coelicolor [69] and Arabidopsis [70]. It will be of interest to learn whether these subunits segregate into homomeric rings, as in E. coli, or whether they combine in heteromeric rings, as in the Rhodococcus 20S proteasome. The ATPases ClpA and ClpX are homohexamers that require nucleotide to be stabilized in the assembled form. The ClpA subunit has two ATPase domains, which, in the hexamer, form two tiers with a cavity between them [71••]. ClpX has a single ATPase domain and forms a single-tiered ring [72]. Specificity
Self-compartmentalization is the principal mechanism whereby proteins that are not specifically targeted for degradation by ClpP or HslV/ClpQ are shielded from their active sites. As with the 20S proteasome, the proteolytic compartment of ClpP is accessible only through a channel that is so narrow (~10 Å) [64] that it may be negotiated only by unfolded polypeptides. Consistent with this scenario, ClpP on its own is capable of degrading peptides with up to about 30 amino acids. The degradation of larger folded protein substrates requires the collusion of an ATPase.
of NSF [55••,56••]. In the D2 hexamer, ATP is bound at the intersubunit interfaces. The absence of cognate sequences in expressed SSD domains of Clp proteases would explain their failure to oligomerize [74••]. In NSF, the binding of ATP by D2 results in oligomerization, whereas D1 — its other ATPase domain — hydrolyses ATP. A similar division of responsibilities has been attributed to the two ATPase domains of ClpA, but in reverse order [78]. In the same vein, the substrate-binding domain of NSF [48••,49••] is at the N terminus, in contrast with the C-terminal disposition of the SSD in ClpA. In the absence of protease, but the presence of nucleotide, the ATPases assemble to their hexameric state and can exercise unfolding or chaperonelike activity [79,80••]. As noted above, fully assembled and active complexes have ATPase rings bound on one or both faces of the protease. Thus positioned, they are strategically placed both to control which proteins have access to the channel leading into the proteolytic chamber and to supervise substrate translocation. In the processing of RepA, the substrate may bind either to ClpA [77••] or to preassembled ClpAP complexes, which can remain associated through multiple cycles of degradation [78]. The orientation of the ATPase rings relative to their proteases is yet to be established. In the case of ClpA, it has been suggested that its C-terminal domain binds to ClpP, on the grounds that this domain is most closely related to ClpX, which also binds ClpP [61]. In this orientation, the distal N-terminal domain would be exposed for substrate binding [77••]. On the other hand, the docking of the C-terminal domains on to ClpP may pose steric problems for ssrAdirected substrate binding by SSD domains, unless they are distributed around, rather than on the face of, the distal ring. Symmetry mismatch
Substrate selection
The presence of multiple energy-dependent proteases in E. coli has been seen as a means of extending the range of substrate specificity, but the system also accommodates some redundancy, as ClpAP, ClpXP and Lon have substrates in common [59,73]. An elegant mechanism has been discovered whereby an 11-residue tag called the ssrA (chromosomal 10Sa gene) becomes appended to the C termini of prematurely terminated proteins. This tag is recognized as a degradation signal by ClpA, ClpX and Lon via homologous sequences in their respective C termini — their so-called SSD (sensor and substrate discrimination) domains [74••]. ssrA tagging does not, however, cover the full range of substrate recognition mechanisms employed by Clp proteases. For certain phage proteins, for example, the λ O protein and the P1 RepA protein, as well as the bacterial SOS response protein UmuD, N-terminal binding has also been documented [75,76,77••]. States of assembly
The closest relative of a Clp ATPase for which a high-resolution crystal structure has been obtained is the D2 domain
An intriguing feature of the ClpAP complex is the discordant symmetries of the rings of the ClpP (sevenfold) and ClpA (sixfold) subunits. As these molecules stack axially, there must be a nonequivalent interaction between the respective rings. A similar mismatch apparently occurs in the proteasome at the interface between the 20S core (definitely sevenfold [14]) and the 19S regulator (putatively sixfold) (see Table 1). Comparable mismatches have been observed in a few other systems — notably, the DNA packaging vertex of bacteriophage capsids and the F1F0 proton transporter. During packaging, the DNA is translocated into the capsid by the terminase (another ATPase) via a connector/portal oligomer embedded in the capsid shell. To date, all connectors that have been convincingly demonstrated to be assembly competent are 12-fold rings that, in their respective capsids, occupy sites of fivefold symmetry. The φ29 connector — the most comprehensively studied such molecule — has been confirmed as a 12-fold ring by high-resolution atomic force microscopy [81] and crystallographic rotation function
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Table 1 Symmetry in subcomplexes of ATP-dependent proteases. Protease subcomplex Eukaryotic 26S proteasome Pseudo 7 Archaeal proteasome–PAN 7 Bacterial proteasome–ARC† 7 Bacterial ClpP–ClpA or –ClpX 7 Human mitochondrial ClpP 7 Bacterial HslV/ClpQ–HslU/ClpY 6 Yeast mitochondrial Lon 7§
Regulator subcomplex Most likely pseudo 6* 6 6 6 ND 6 and 7‡ 7§
*Six proteasomal ATPase genes are found in the yeast genome. † An interaction between ARC and the proteasome has not been demonstrated yet. ‡ Overexpressed HslU forms sixfold and sevenfold symmetric particles. §Protease and ATPase domains are linked on a single polypeptide chain. ND, not determined.
analysis [82]. Alternatively, in one case (bacteriophage SPP1), overexpressed connectors have been reported to be 13-fold rings [83] and, in another, bacteriophage T7, to be a mixture of 12-fold and 13-fold rings [84]. It is not yet clear whether the 13-mers are assembly competent: if not, their order of symmetry most likely represents an artifactual consequence of overexpression. Another such mismatch occurs within F1F0 [85], in which the pseudo-threefold F1-ATPase overlies the intramembranous F0 component, which has a much higher order of symmetry (10- or 12-fold) [86,87]. In both systems, the mismatch has been implicated in relative rotational movement [88,89] and such rotation has been elegantly demonstrated in the F1F0 system [90,91]. Returning to energy-dependent proteases, it is appealing to equate the symmetry mismatch with the relative rotation of the ATPase and the protease during processive Figure 2 Axial view of the ClpAP complex at 29 Å resolution [71••]. ClpP (yellow) has sevenfold symmetry and ClpA (blue) has sixfold symmetry, so that the interactions between pairs of subunits in the two rings are nonequivalent. Their binding is inferred to be dependent on a key interaction (white asterisk) between one specific subunit in ClpA (red dot) and one ClpP subunit (green dot). Relative rotation by only 8.6° (equivalent to a shift of 4.5 Å at the point of contact) transfers the key relationship to neighboring subunits. Thus, the symmetry mismatch is conducive to relative rotation without disengagement of the two subcomplexes. Such rotation might occur during the processive translocation of substrates into ClpP [71••]. A similar symmetry mismatch occurs in the 26S proteasome and some other energy-dependent proteases (see Table 1).
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digestion of substrates. In the ClpAP system, the relative angular disposition of the ClpP and ClpA rings has been determined [71••]. This interaction is based on key subunits in both rings. Key status is transferred to neighboring subunits by a small rotation (8°), corresponding to a translational shift of only 4–5 Å at the point of contact (Figure 2), a property that facilitates rotation without separation of the subcomplexes. On the other hand, HslVU/ClpQY complexes assembled in vitro exhibit 6:6, as well as 6:7, symmetry combinations ([92,93]; see Table 1). Lon, whose ATPase and protease domains are covalently linked, inevitably requires equimolarity in active complexes. For questions pertaining to the functional implications of symmetry mismatches for processive, energy-dependent proteolysis, final answers are unlikely to precede further experiments.
Conclusions Common to all known energy-dependent proteases is a linear arrangement of the proteolytic modules and the regulatory modules, which prepare substrates for degradation. These tasks include substrate recognition, unfolding and translocation. Although the exact role of ATP hydrolysis remains to be established, it is likely that the energy-dependent steps are unfolding and translocation. Although not universal, a recurring feature is a symmetry mismatch between the ATPase module and the proteolytic module. This property, which occurs in the proteasome, as well as in the ClpAP and ClpXP proteases, has led to the hypothesis that the symmetry mismatch may facilitate relative rotation of the respective modules in the processive degradation of substrates. However, there is no such mismatch in Lon nor, putatively, in ClpYQ (HslUV) [94••]. This may indicate that processivity in the latter complexes is effected by movements involving axial translations, rather than relative motions between the respective modules.
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Acknowledgements We thank Sorel Fitz-Gibbon and Andreas Ruepp for communication of unpublished results and David Belnap and Kornelius Zeth for help with the graphics.
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