ec

e, L

Reviewproteasome core particle (CP), where substrate proteolysisoccurs, and the 19S regulatory particle (RP) complex,which recognizes and unfolds Ub-tagged substrates.

The CP can be likened to a nonspecific shredder, with acylindrical barrel-like architecture composed of four stackedheptameric rings, each comprising a- and b-type subunitsarranged in abba stoichiometry (Figure 1) [35]. Simplerorganisms such as archaebacteria have CPs with 14 iden-tical a-subunits and 14 identical b-subunits, whereas morecomplex organisms such as eukaryotes have seven differenta- and seven different b-subunits that form an a17b17b17a17 particle. Structure comparison revealed that a and bsubunits share a similar fold: a four-layer structure inwhich

Biogenesis of the archaeal 20S proteasome appears to occurinan ordered set of steps startingwith the formation of thearing, which acts as a platform onto which b subunits assem-ble, thus forming a half-proteasome that subsequentlymatures into the CP. In most prokaryotes, a subunits havethe ability to spontaneously self-assemble, forming a seven-membered homo-oligomeric ring [14]. This unique propertystems from the fact that neighbouring a-subunits possess acharacteristic loop, known as the L-loop, which facilitates aconspicuous contact surface interaction between these sub-units [3]. By contrast, b-subunits lack the L-loop, and thuscan only assemble using the a-ring as a platform [15].Moreover,b-subunits are genetically expressed in an imma-ture precursor form, thereby preventing uncontrolledself-assembly of the catalytic subunits and active siteCorresponding author: Groll, M. (michael.groll@ch.tum.de).The 26S proteasomfunction of a destruNerea Gallastegui and Michael Groll

Center for Integrated Protein Science at the Department ChemiLichtenbergstr. 4, 85747 Garching, Germany

The heart of the ubiquitin-mediated degradation path-way, the 26S proteasome, endoproteolytically cleavesmost intracellular proteins, thereby maintaining bio-logical homeostasis and regulating many crucial pro-cesses in the cell. This hydrolyzing machine comprisesmore than 30 different subunits, which perform differentfunctions including the recognition, unfolding, translo-cating and cleavage of protein substrates. Thus, carefulassemblage and regulation of the 26S proteasome isessential to ensure correct positioning and function ofeach subunit, thereby preserving the delicate cellularbalance between protein synthesis and degradation.Here, we review the most current research on the 26Sproteasome assembly pathway, and describe the mech-anism used by the cell to manage the complex structureand functions of the proteasome.

The 26S proteasome and its involvement in theubiquitinproteasome system (UPS)Protein synthesis and protein degradation are importantprocesses in cellular homeostasis that ensure maintenanceof protein regulation. The main non-lysosomal proteindegradation pathway used for this purpose is the UPS[1]. Substrate degradation by the UPS is rigorously con-trolled by the carboxyterminal tagging of the 76-residueprotein ubiquitin (Ub) to surface-exposed lysine residues ofredundant or misfolded proteins. Once the initial Ub isattached, polyubiquitylation of the substrate occursthrough the sequential transfer of additional Ub mol-ecules, forming Ub chains. These chains are recognisedby the proteolytic core engine of the UPS, the 26S protea-some, which degrades the substrates of interest intodefined oligopeptides. This sophisticated multicatalyticalcomplex [2] is divided into two main structures: the 20S634 0968-0004/$ see front matter 2010 Elsevier Ltd. All rights reserved. doi:10: assembly andtive machine

ehrstuhl fur Biochemie, Technische Universitat Munchen,

two sheets of antiparallelb strandsare sandwichedbetweentwo layers of a helices [3]. Substrate peptide bonds arehydrolysed by the N-terminal threonine residue, Thr1,which is embedded in the b subunits [6]. Interestingly,whereas each b subunit is active in simpler organisms,thereby providing 14 equivalent catalytic sites per CP,eukaryotic substrate degradation is more specific [7],although only three of the seven different b subunits areproteolytically active [4]. Hence, the network between theinactive and the active eukaryotic b-type subunits shapesunique substrate binding channels, giving rise to threedistinct active sites located in the b1, b2 and b5 subunits,with specific cleavage preferences known as caspase-, tryp-sin- and chymotrypsin-like activities, respectively [8]. Thiscomplicated architecture justifies the great amount ofenergy consumed by the cell to assemble this moleculardegradation machinery correctly. Furthermore, the similartopologies, structuresandmechanismsofCPs fromdifferentorganisms suggest that the proteasomes not only originatefrom an ancestral gene, but that nature has, through evol-ution, optimised and adjusted this complex to meet themultifaceted demands of higher organisms. Understandinghow such complex machinery assembles is of vital import-ance to comprehend fully the overall regulation of proteol-ysis in the cell. In this review,weaim todescribeprokaryoticproteasome assembly and discuss how eukaryotes havetaken advantage of several intracellular proteins to copewith the increased complexity in their CP and RP assemblypathways [913]. Indeed, the cell must not only avoid non-specific substrate cleavage, but also ensure the correctpositioning of each 26S proteasome subunit, thereby form-ing a functionally active particle.

Prokaryotic CP assembly.1016/j.tibs.2010.05.005 Trends in Biochemical Sciences, November 2010, Vol. 35, No. 11

itec

llust

diff

s a

easoexposure [16]. The final step in CP assembly is the inter-action between two immature half-proteasomes, whichensures the autocatalytic processing of the propeptidesand exposure of the active sites into a closed, mature 20Sproteasome [17].

Eukaryotic CP assembly: a-ring formationAssembly of eukaryotic CPs follows a very similar pathwayto that described for archaeal proteasomes, but its morecomplex architecture requires the involvement of dedi-cated assembly factors [11]. Protein chaperones such asproteasome-assembling chaperones (PAC)14 [1820] andmaturation factors including ubiquitin maturation protein(UMP)1 [21] direct the proper positioning of the different aand b subunits, thus leading to successful 20S particle

Figure 1. The proteasome.

Crystal structure of the 20S proteasome CP showing its cylindrical barrel-like arch

subunits arranged in abba stoichiometry [35]. (a) Most archaebacterial CPs, as i

identical b-subunits, whereas (b) eukaryotic CPs have seven different a- and seven

dependently regulated by the 19S regulatory complex (RP) (c), here represented a

unfolds and translocates Ub-tagged substrates into the active sites of the 20S prot

Reviewmaturation.PAC1PAC2 and PAC3PAC4 are the two main hetero-

dimeric chaperone complexes involved in a-ring assembly.The PAC1PAC2 complex binds the a5 and a7 subunits,and assists a-ring formation by preventing undesirable,spontaneous a-subunit dimerisation [22]. Interestingly,the half-life of the PAC1PAC2 complex is consistent withthe estimated maturation period of the eukaryotic CP,indicating a direct correlation between PAC1PAC2 diges-tion and active 20S proteasome formation. This findingalso suggests that the chaperone complex is bound to theCP throughout most of the assembly pathway [22].Whether PAC1PAC2 is located at the surface or in thecentre of the immature 20S proteasome is currently acontentious issue [10]. However, its surface location couldlead to an additional role as a protector of the eukaryotic20S proteasome entrance pore, thus preventing nonspecificcleavage of the surface-exposed unstructured N-terminaltails of the various a-subunits during particle assembly.

Recently, another chaperone complex intermediate wasidentified in association with immature CPs: the PAC3PAC4 complex [23,24]. This heterodimer has only beendetected in the early steps of proteasome maturation,and differs in both function and structure from thePAC1PAC2 complex [19]. The primary role of thePAC3PAC4 complex is to inhibit the inappropriate bind-ing of a-subunits by proof-reading the arrangement ofindividual a subunits, thereby maintaining the hierarchybetween a-subunit incorporation during ring assembly[24]. This idea was confirmed by the detection of defectivea-ring structures in yeast mutants lacking the PAC3 andPAC4 chaperone homologues [23,24]. Interestingly, thestructure and topology of the PAC3PAC4 complexresembles that of proteasomal subunits. Thus, the chaper-one could possibly act as a wild card, so that subsequentdisplacement of the PAC3PAC4 complex occurs only bythe addition of the b-subunit with higher binding affinity.Finally, release of the PAC3PAC4 complex only occursonce the a-ring has been formed and the b-subunits are

ture composed of four stacked heptameric rings, each comprising a- and b-type

rated for Thermoplasma acidophilum [3], contain 14 identical a-subunits and 14

erent b-subunits that form an a17b17b17a17 particle [4]. Eukaryotic CPs are ATP-

cartoon, reflecting the lack of high-resolution structural data. The RP recognizes,

me.

Trends in Biochemical Sciences Vol.35 No.11being incorporated. This suggests that the transient and/orweak interaction between PAC3PAC4 and the b-subunitstriggers chaperone disassembly [24].

Eukaryotic b-ring formation and half-proteasomeassemblyThe crystal structure of the mature yeast 20S proteasomeshows that each b-subunit has a unique set of amino acidsthat specifically interact with its neighbouring subunits,forming a complicated interdigitating network throughoutthe entire b-ring [4]. A prime example of these intermole-cular interactions is found in subunits b2 and b3, in whichthe C-terminus of the b2 subunit embraces the b3 subunitby forming an anti-parallel b-sheet that also interacts withsubunit b4 andwith subunits b60 and b70 from the adjacentb0-ring. Hence, the correct assembly of such a complexnetwork in eukaryotic cells requires additional mechan-isms and aiding peptides for precise b-subunit incorpora-tion [21]. The b-subunit precursor peptides (b-propeptides)are crucial for b-ring assembly. The b5-propeptide, forexample, plays an important role in the maturation path-way and is crucial for cell survival; its chromosomaldeletion leads to impaired proteasome assembly and, con-sequentially, cell lethality [16]. Interestingly, when cells

635

co-express the b5-propeptide in trans they restore wild-type like growth [16]. Therefore, the function of theb5-propeptide is similar to that of chaperones, in the sensethat it is crucial for 20S particle maturation but itself is notpart of the final complex. Moreover, the b5-propeptide isalso required for the specific interactions between theb-subunit and an additional chaperone, UMP1 [21]. Never-theless, it still needs to be determined whether this inter-action is unique to the b5-propeptide or if it also occursbetween UMP1 and other b-precursors.

UMP1 was the first chaperone shown to be part of aproteasomal precursor complex containing both the a-ringand unprocessed b-subunits, thus suggesting its involve-ment in recruiting b subunits during CP maturation [21].Small interfering (si)RNA-mediated knockdown of eachb-subunit in mammalian cells revealed that incorporationof the various b-subunits into the a-platform follows aprecise mechanism, and established b2 as the first subunitdocked onto the a-ring [25]. Additionally, UMP1 promotesthe proper entry order of subunits b3 to b6. Subunit b7 isthe final b-subunit integrated into the ring, hence forminga half 20S proteasome known as the 15S complex [26](a17b17UMP1PAC1PAC2) [25]. Interestingly, theC-terminal tail of subunit b7 possesses a unique chaper-oning function in the final CP maturation step. Yeast cellsharbouring a mutant version of b7 that lacks its C-termi-nus show an accumulation of half-proteasomes that

include not only a- and b-subunits, but also UMP1 andPAC1PAC2 [27]. These findings are in agreement withthe crystal structure of the mature yeast 20S proteasomein which the b7-C-terminus extends from one b-ring to theadjacent b0-ring, closely interacting with subunits b10 andb20 4. However, subunit b7 is not the only one responsiblefor the correct orientation of the two half-proteasomes;specific interactions between distinct sequence motifs inthe b and b0 subunits also contribute to the final mature20S particle (Figure 2). Finally, CP assembly is completedby maturation of the sequestered proteolytically activesites. This step is accomplished by intramolecular auto-lysis of the propeptides of the proteolytically activesubunits b1, b2 and b5, an event which requires a Gly(-1)-Thr1 sequence motif as well as both b- and b0-rings.The result of this process is the full activation of thecatalytic site [17]. Of note, this final maturation step alsoincludes the partial processing of propeptides from inactiveb-subunits [28,29]. Once the assembly has been completed,the proteasome liberates its newly formed cavities throughthe degradation of PAC12 and UMP1 (Figure 2) [21,22].The catalytic sites are mature, and therefore free accessinto the CP must be blocked to protect the cell fromunregulated protein degradation. This role is undertakenby the N-termini of the a-subunits, which form a gate thatcan only be opened in a strictly regulated manner throughthe binding of regulatory particles [30,31].

occ

bun

e the

he l

atur

Review Trends in Biochemical Sciences Vol.35 No.11Figure 2. 20S proteasome assembly

The current model for CP assembly proposes the following steps: (i) a-ring formation

UMP1 and the b2 subunit dock onto the a-ring [25]; (iii) UMP1 promotes proper b-su

proteasome structure; (iv) two half-proteasomes subsequently dimerise and activat

20S proteasome; (v) PAC1PAC2 complexes are either degraded or released from t

and propeptides of the active b-subunits that are released by autolysis during CP mby the b-propeptides (which directly block the proteolytic active sites), whereas inhi

condense to form a closed proteolytically active chamber [19,21].

636urs with the aid of two heterodimeric chaperones, PAC1PAC2 and PAC3PAC4; (ii)

it entry order in the ring system and displaces PAC3PAC4, thereby forming a half-

proteolytic sites via autolysis, and encapsulated UMP1 is degraded by the mature

atent CP. Inhibitory N-terminal sequences of a-subunits forming the entrance gate

ation are represented in red. In the inactive half-proteasome, inhibition is providedbition by the a-subunit tails becomes effective only when the half-proteasomes

tory

he y

ome

the

gat

olec

tant

esid

at th

yotiOpening doors into the 20S proteasomeThe structural organization of the 20S proteasome imposesstrong constraints on the access of substrates into theproteolytic chamber and the release of degradation pro-ducts [4,5]. In archaeal proteasomes, two entry ports ofabout 13 A in diameter are located in the centre of the a-ring, at both ends of the CP [3,15]. By contrast, the hydro-lytic chamber of the yeast and bovine 20S proteasomes aretightly sealed, as the N-terminus of each individuala-subunit projects into the pore of the a-ring, formingseveral layers. Thus, access into the interior cavity ofthe eukaryotic CP requires substantial structural re-arrangements. Surprisingly, the catalytic activation ofthe CP is dependent on a simple sequence motif (theYDRmotif, with the amino acids a3-Asp9 hydrogen bonded

Fig. 3. Gate opening for substrate accessibility into the 20S proteasome.

Substrate accessibility into the hydrolytic chamber of the CP is restricted by a regula

(RP). (a) Gate opening mechanism triggered by RP binding to the 20S proteasome. T

the a-subunits (shown in red), which are structurally rearranged in the 26S proteas

calpain inhibitor I [4] (yellow), which marks the distinct proteolytically active sites of

the yeast 20S proteasome (yellow box). The right panel shows a close-up view of the

position 13, are drawn in specific color coding, whereas the remaining part of the m

the yeast CP. Deletion of the first 12 N-terminal amino acids of subunit a3 (a3DN-mu

subunit a3 plays a key role in gate formation (black line). Mutagenesis within this r

mutant. Structural rearrangement of the gate does not produce an allosteric effect

levels similar to that reported for the 26S proteasome. Thus, turnover of the eukar

chamber of the CP. Part of this figure has been previously published [30].

Reviewwith a4-Tyr8 and a4-Arg10), which is conserved among alleukaryotes [30]. Amino acid substitution of the aspartateto an alanine in the YDR motif leads to a conformationalchange of all a-N-termini. This structural rearrangementdoes not produce an allosteric effect in the proteolytic sites,but does significantly enhance peptidase activity to levelssimilar to those reported for the 26S proteasome [30].Binding of the 19S regulatory particle to the 20S protea-some triggers the structural rearrangement of the atailsegments (Figure 3) [32]. This indicates that the closedstate of the channel found in the crystal structure of thewild-type yeast CP is the predominant conformation insolution [4,30], thus forming a latent 20S proteasome.Opening of the gate in CPs unwraps a narrow restrictedchannel into the a-ring of about 13 A in diameter, ensuringthat protein degradation occurs only if the substrate isunfolded, a task undertaken in the cell by the 19S RP.

Overall structure of the 19S regulatory particleThe 19S regulatory particle is a multi-subunit complexfound in eukaryotes that not only is involved in the recog-nition, unfolding and translocation of ubiquitylated sub-strates, but also regulates the entry of selected substratesinto the core particle [2]. Biochemical and structuralstudies have subdivided the RP into two main structures:the base and lid [33]. The base is composed of six differentAAA (ATPases associated with a variety of cellular activi-ties) -type ATPase subunits[34], which are commonlyknown as (regulatory particle triple A proteins (Rpt)16,and by three non-ATPase subunits known as regulatoryparticle non-ATPase proteins (Rpn)1, Rpn2 and Rpn13[33]. The Rpts share high sequence homology, thussuggesting that the domains share common folds andtopologies. The lid complex consists of nine subunits(Rpn3, Rpn59, Rpn1112 and Rpn15), all with uniqueprimary sequences, structures and functions. The base andthe lid are connected to each other by the linker subunitRpn10, thereby forming the 19S regulatory particlecomplex [33].

gate, which is unlocked upon ATP-dependent binding of the 19S regulatory particle

ellow box emphasises the entrance pore formed by the N-terminal tail segments of

. (b) Transverse view of the surface representation of the yeast CP in complex with

CP. The closed substrate entry pore is observed in the wild-type crystal structure of

e, formed by the N-terminal tails of the a-subunits. These N-terminal residues, up to

ule is in grey. The closed conformation is seen in the wild-type crystal structure of

) causes a locked stage of the open gate conformation in the CP [30]. Note, Asp9 of

ue generates an axial channel whose dimensions are identical to that of the a3DN

e proteolytic sites; however, it significantly enhances peptidase activity, reaching

c CP is strictly dependent on the substrate accessibility into the central hydrolytic

Trends in Biochemical Sciences Vol.35 No.11Both the spatial organization of each 19S subunit andtheir defined roles in the 26S holocomplex are currentlyunknown, thus making it difficult to allocate individualfunctions to the base and the lid. Originally, the base wasthought to be involved in substrate unfolding and translo-cation and the lid in substrate recognition and Ub release.More recent results have shown that yeast base subunitssuch asRpt5 andRpn13, and the linker subunit Rpn10, canbind ubiquitin chains [3539]. It is therefore not surprisingthat elucidating the structure and assembly pathway of the19S regulator is of great scientific interest.

Gate opening of the 20S proteasome by the 19Sregulatory particleAlthough studies of the archaeal CP regulatory particle,proteasome activating nucleotidase (PAN) (Box 1), haveresulted in important insights into the mechanism andstructure of proteasome regulation, many questions on theeukaryotic RP still remain unanswered owing to its highdegree of complexity. Furthermore, studies on the inter-action of both PAN and the 19S ATPases with the 20Sproteasome have proven difficult because of the symmetrymismatch between their hexameric ring structure andthe heptameric 20S proteasome [40]. However, this

637

Box 1. ATP-dependent regulation in prokaryotes

The lack of Ub in prokaryotes initially led to the assumption thatproteasomal regulatory particles were present only in eukaryotes.However, an interesting association between prokaryotic andeukaryotic proteasomal regulation arose through the discovery ofan ATPase complex found in many archaea, which shares 40%sequence identity with the six RPTs of the eukaryotic base [68]. Thisdonut-shaped homo-hexameric complex, termed proteasome-acti-vating nucleotidase (PAN), could therefore be the evolutionaryancestor of the RP before coupling of proteasomal function withubiquitination [6971]. Interestingly, as observed in eukaryotic CPs[32], activation of the archaeal CP takes place by transientassociation of PAN to its 20S proteasome via ATP binding [69,71].The architecture of PAN can be divided into two substructures [7274]: subcomplex (SUB) I, which is responsible for target proteinrecognition and formation of the entry port of the substratetranslocation channel, and SUB II, which comprises the ATPase-domains for substrate protein unfolding and associates with the CP.It is the synergistic combination of SUB I and SUB II that allowssubstrate recognition, unfolding and translocation, as confirmed bymutational studies in the linker segment between these twosubcomplexes [73,75]. Furthermore, structural and functional dataproved that ATP-dependent proteasome regulation is organized in aseries of well defined subsequent steps: (i) RPs are associated withthe CP, (ii) opening of the substrate entry channel into the CP, (iii)binding of a defined set of substrates, (iv) global unfolding of thesubstrates on the surface of the ATPase, and (v) processivetranslocation of the unfolded substrate into the lumen of the CP

Reviewdiscrepancy might allow multiple conformations and thusprovide the flexibility required for triggering the structuralrearrangements necessary for opening the entrance pore ofthe CP [40,41]. The complications provided by this mol-ecular flexibility havemeant that there is currently no highresolution data available. Nevertheless, structural modelsof the archaeal RP and functional studies on the eukaryoticRP have led to the first mechanistic insights into theproteasome regulators [4244]. Site-directed mutagen-esis-mediated inactivation of nucleotide binding in thedistinct ATPase domains of each Rpt-subunit revealedstable and properly matured 26S proteasomes [32]. How-ever, the Rpt2 mutant, which results in a closed 26Sholoenzyme conformation, displays peptidase activitysimilar to that observed in the latent CP [45]. Interest-ingly, the Rpt2 mutant regains fully active and stable 26Scomplexes when co-expressed with the CP open gatemutant, therefore confirming that it is indeed the bindingand/or hydrolysis of ATP in Rpt2 that triggers the confor-mational changes of the a-N-terminal tails in the CP.Recent experiments have demonstrated that it is not onlythe binding of ATP to Rpt2 that opens the 20S gate, butalso the binding of the C-terminal tails of Rpt1, Rpt2 andRpt5 to theCP [43]. These Rpts share a conserved sequencemotif at their C-termini, consisting of a hydrophobic amino

[43,47,76,77]. Interestingly, the latter step is exerted mainly by ashort conserved segment motif located in the core of the centralchannel. This short motif, found in most AAA-type ATPases, iscomposed of an aromatic residue followed by a hydrophobic one(ArF loop) and acts as the final checkpoint and rate-limiting step foraccessibility of unfolded substrates, by exerting a powerful inwardstroke movement dragging the substrate into the CP [78]. Thesefindings and the sequence conservation shared between the variousdomains of PAN and the Rpt subunits of the eukaryotic basecomplex suggest a common function of ATP-dependent protea-some regulation, and open a small window towards the completeunderstanding of these unfoldases.

638acid followed by a tyrosine and a final residue X (HbYXmotif), which is necessary for gate opening [46,47].

Interactions between different Rpt-C-terminal seg-ments and the CP were further characterised by replacingthe PAN C-terminal tail residues with the correspondingeukaryotic C-termini of Rpt1Rpt6 [43]. Studies on theeffects of these PANRptmutants on the yeast a-ring showedthat the C-termini of subunits Rpt2 and Rpt5 play a keyrole in modulating entry pore status in eukaryotes [46].Furthermore, electron micrographic (EM) single particleanalysis identified a combination of interactions betweenthe PAN-HbYX motif and distinct amino acids of each a-subunit that resulted in an induced-fit conformationalchange of the CP regulatory gate: (i) the carboxyl groupof the HbYX terminus forms a salt bridge with the e-NH2group of Lys66 of the corresponding a-subunit; (ii) the Tyr-OH side chain in the HbYX motif is hydrogen bound withthe carbonyl oxygen of Gly19 of a-subunits and (iii) the Hbin the HbYX motif forms van der Waals interactions withthe intermolecular hydrophobic pocket of two adjacenta-subunits [43]. Although the results of the EM-studiesdid not explain the symmetry mismatch between thehexameric and pseudohexameric structures of PAN andRpt16 and their corresponding heptameric CPs, they didprovide the first insights into the interactions between theHbYX-motif of the Rpts and eukaryotic CPs. Surprisingly,the characteristic tail sequences are only found in subunitsRpt2, Rpt3 and Rpt5, with the C-terminus of Rpt5 beingthe most potent stimulator for gate opening [43,46],whereas amino acid substitutions in the nucleotide bindingmotif in subunit Rpt2 strongly repress substrate accessi-bility into the CP [32]. Thus, the concerted structuralrearrangement of the Rpt2-ATPase domain and thedistinct Rpt-HbYX C-termini are believed to jointly causea conformational change on the N-terminal tails ofCP-subunits a3 and a4 [4,5]. As mentioned, Rpt1, Rpt4and Rpt6 lack the HbYX motif, but interestingly, theC-termini of subunits Rpt4 and Rpt6 are essential forthe binding of external chaperones that facilitate orderedassembly of the RP [12].

Assembly of the 19S regulatory base complexTodate, four different external proteins playing a key role in19S base assembly have been identified: Nas2, Nas6, Hsm3and Rpn14, collectively known as 19S-specific assemblyfactors [48]. Surprisingly, althougheachof these chaperonesbinds the C-termini of Rpts, they possess unique architec-tures and folds, thus implying converged evolution of theirfunction in RPmaturation. Although 19S-specific assemblyfactors were originally considered to be proteasome subu-nits, none of these factors are present in the mature 26Sproteasome. The high binding affinity to free RP subunits,along with the defect in base assembly that occurs in theirabsence, led to the suggestion that these factorsmight serveas chaperones in the 19S maturation pathway. Each 19Sassembly factor specifically binds to a defined Rpt subunit,resulting in the formation of four initial pairs of complexes:Rpt1Hsm3, Rpt3Nas6, Rpt5Nas2 and Rpt6Rpn14[4853] These precursors subsequently assemble into

Trends in Biochemical Sciences Vol.35 No.11three stable intermediates and recruit additional base-sub-units: Rpn1Rpt2Rpt1Hsm3, Rpt4Rpt5Nas2 and

ReviewNas6Rpt3Rpt6Rpn14; the latter contains two 19S-specific assembly factors and might act as the seed informing the base. Subsequently, the three precursormodules bind Rpn2 and Rpn13 to jointly complete assemblyof the base, which then attaches to the lid via the Rpn10linker subunit [48,52] (Figure 4). The mechanism, bindingproperties, and release of the 19S-specific assembly factorsfrom the RP complex remain a matter of debate; however,recent work indicates that the C-terminus of Rpt6 isinvolved in the disassembly of Nas2 and Hsm3 [12,52].These two chaperones associate with the RP only duringbase maturation and are displaced upon incorporation ofsubunits forming the lid complex as observed for Rpn14. Bycontrast, Nas6 is attached to the RP until maturation of the19S complex is complete, thus representing one of the finalchaperones to be released from the 26S proteasome [48].These observations indicate thatNas6might coordinate thecomplex formation of the RP to the CP. Thus, Nas6 mightprotect the surface-exposed C-termini of the distinct Rptsfrom nonspecific proteolytic cleavage upon proteasomematuration. Additional functional and structural exper-iments are required to gain a clearer understanding ofthe mechanism of assembly and disassembly of the 19S-specific assembly factors and of RP maturation. Of note,there is evidence for an alternative maturation pathway inwhich theCPactsas thenucleus for 19Sassembly [12,54]. In

Figure 4. Assembly and structure of the 19S regulatory particle

The 19S base assembly and postulated subunit interactions of the lid. RP base assembl

Rpn1Rpt2Rpt1Hsm3, Nas2Rpt3Rpt6Rpn14 and Nas6Rpt5Rpt4; (ii) the three init

Hsm3, Rpn14, Nas6 are released through the addition of Rpn10 and the lid; (iv) Nas2 is

assembly [48,52]. Nas6 is the first chaperone to be displaced, whereas Hsm3 and Rpn14

for an alternative pathway, it is not shown in this figure [85,54].Trends in Biochemical Sciences Vol.35 No.11this model, the 19S subunits Rpt2, Rpt4, Rpt6 and possiblyRpt3 assemble first on the 20S proteasome and mediatebinding of the Hsm3Rpt1Rpt2Rpn1Rpt5 complex. Inthis way, Rpn14, Nas2 and Nas6 are expected to interactwith the C-terminus of the 19S subunits with the uniquepurpose of preventing gate opening of the CP [13]. However,more knowledge of the structures and interactions of thesesubunits within the 19S base are necessary to understandthe function of these assembly factors and sequence ofevents within this pathway.

Assembly of the lid complexAt present, little is known about the composition of the 19Slid complex. However, its structural relation to that ofeukaryotic translation initiation factor (eIF)3, a multicom-ponent factor required for the initiation of protein biosyn-thesis, and of the constitutive photomorphogenesis (COP)9complex, an essential regulator of diverse cellular anddevelopmental processes [55], has led to the first insightsinto its architecture. All three complexes contain at leasteight different core subunits, with a remarkable one to onesequence correspondence to each other that suggests acommon ancestor [33]. To date, the lid complex is believedto be formed through two main sets of subunits: Rpn5Rpn6Rpn8Rpn9 and Rpn3Rpn7Rpn15, which sub-sequently interact with Rpn11 and Rpn12 [56,57]

y takes place in a series of steps: (i) RP base subunits form three initial complexes,

ial complexes subsequently assemble into the base complex; (iii) the chaperones

finally displaced through CP binding; (v) plausible chaperone detachment in 19S

disassemble with the help of the Rpt6 C-terminus. Note, although evidence exists

639

(Figure 4). Although assembly of the lid subcomplexappears to occur independently from that of the base, itscomplexity suggests that additional chaperones arerequired for correct maturation. For example, heat shockprotein (Hsp)90 is known to be involved in lid formation, asits deletion causes impaired assembly [58]; the existence ofadditional specific lid-dedicated chaperones still remainsto be confirmed.

Concluding remarks and future perspectivesIn this review, we have presented the latest discoveries inproteasome structure, function and assembly. As dis-cussed, complex organisms such as eukaryotes, whichharbour elaborate proteasome structures, have developedexternal help from chaperones to cope with its complexity.Simpler organisms, such as bacteria, have a less sophisti-cated structure and so can autonomously self-assemble.However, it must be mentioned that prokaryotic protea-somes also undergo alternative assembly pathways, inwhich a and b subunits form initial dimeric complexesbefore ring formation [59] (Box 2). Nevertheless, eventhough the prokaryotic assembly pathway has been wellcharacterized, further studies in the eukaryotic protea-some are necessary to obtain a much clearer picture ofits assembly. Additional proteins such as Ecm29 [60] orBlm10 [61,62] have been shown to bind to the proteasome.These proteins could also be involved in the proteasomalassembly pathway [63]; however, their precise roleremains controversial. Ecm29 can tether the CP to the

ReviewRP, whereas Blm10 stabilises the pre-holo-CP and surpris-

Box 2. Alternative pathways of CP assembly and regulation

The maturation pathway of bacterial CPs differs significantly fromthat of archaea and eukaryotes. In bacteria, 20S proteasomes arefound only in actinomycetes [59]. In these CPs, adjacent a-subunitshave a much smaller surface contact area that prevents sponta-neous ring formation. Therefore, to enable further particle assem-bly, the b-propeptides act as assembly-promoting factors by linkingtheir own b-subunits to two adjacent a-subunits. However, mostbacteria only contain a minimal proteasome, such as the HslUVsystem [79], in which HslV acts as the protease that is regulated byHslU in an ATP-dependent manner. Although self-compartmentali-zation is a generally conserved principle in all CPs, the architectureof HslV represents a simpler version of 20S proteasomes, consistingof only two homo-oligomeric rings [80]. HslV protomers arestructurally and functionally homologous to CP b-type subunitsand spontaneously self-assemble without any additional help frompropeptides and chaperones. Whereas archaeal and eukaryotic CPsare formed by seven-membered rings, the bacterial HslV-ringconsists of only six subunits, with absence of the C-terminal helix[80]. Nevertheless, the hexameric double ring-system of HslVenfolds a large cavity that houses the active sites and thusconserves the proteolytic mechanism as observed for 20S protea-somes [8]. Interestingly, similar to most archaeal and eukaryoticCPs, HslV alone only has minor peptidase activity and is ATP-dependently regulated by HslU [8183]. However, the activationmechanism of eubacterial HslV is allosteric and therefore uniqueamong CPs [84]. Thus, proper peptide bond cleavage in the HslUVsystem is facilitated by an induced conformational change in theactive site region of the protease. The C-termini of Hs1U binds intotwo pockets between adjacent subunits of HslV and displaces theirapical helices, thereby causing structural reorganisation of thesubstrate-binding channel in its core particle. Therefore, HslVreveals a fundamentally different binding mechanism from that of

the eukaryotic and archael 20S proteasomes.

640ingly has much higher affinity to the open gate CP than tothe latent CP [64]. How these proteins affect the assemblyof the 26S proteasome remains controversial, and is anarea of research that offers extensive room for new andexciting discoveries. Nevertheless, these recent insightsinto 26S proteasome function and maturation present astep forward towards unravelling its sophisticated assem-bly puzzle. They will also open new and exciting leads fordrug design that might regulate proteasome levels in thecell, contributing to therapeutic interventions of cancer[65], neurodegenerative disease [66] and immunologicaldisease [67].

AcknowledgmentsWe thank Wolfgang Heinemeyer and Martin Cullell-Young for helpfuldiscussions.

References1 Hershko, A. and Ciechanover, A. (1998) The ubiquitin system. Annu.Rev. Biochem. 67, 425479

2 Voges, D. et al. (1999) The 26S proteasome: a molecular machinedesigned for controlled proteolysis.Annu. Rev. Biochem. 68, 10151068

3 Lowe, J. et al. (1995) Crystal structure of the 20S proteasome from thearchaeon T. acidophilum at 3.4 A resolution. Science 268, 533539

4 Groll, M. et al. (1997) Structure of 20S proteasome from yeast at 2.4 Aresolution. Nature 386, 463471

5 Unno, M. et al. (2002) The structure of the mammalian 20S proteasomeat 2.75 A resolution. Structure 10 (5), 609618

6 Seemuller, E. et al. (1995) Proteasome from Thermoplasmaacidophilum: a threonine protease. Science 268, 579582

7 Coux, O. et al. (1996) Structure and functions of the 20S and 26Sproteasomes. Annu. Rev. Biochem. 65, 801847

8 Groll, M. et al. (2005) Molecular machines for protein degradation.Chem. Bio. Chem. 6, 222256

9 Marques, A.J. et al. (2009) Catalytic mechanism and assembly of theproteasome. Chem. Rev. 109, 15091536

10 Murata, S. et al. (2009) Molecular mechanisms of proteasomeassembly. Nat. Rev. Mol. Cell Biol. 10, 104115

11 Ramos, P.C. and Dohmen, R.J. (2008) PACemakers of proteasome coreparticle assembly. Structure 16, 12961304

12 Park, S. et al. (2010) Assembly manual for the proteasome regulatoryparticle: the first draft. Biochem. Soc. Trans. 38, 613

13 Besche, H.C. et al. (2009) Getting to first base in proteasome assembly.Cell 138, 2528

14 Zwickl, P. et al. (1992) Expression of functional Thermoplasmaacidophilum proteasomes in Escherichia coli. FEBS Lett. 312, 157160

15 Groll, M. et al. (2003) Investigations on the maturation and regulationof archaebacterial proteasomes. J. Mol. Biol. 327, 7583

16 Chen, P. and Hochstrasser, M. (1996) Autocatalytic subunit processingcouples active site formation in the 20S proteasome to completion ofassembly. Cell 86, 961972

17 Ditzel, L. et al. (1998) Conformational constraints for protein self-cleavage in the proteasome. J. Mol. Biol. 279, 11871191

18 Davies, A.M. et al. (2005) Crystal structure of a putativephosphinothricin acetyltransferase (PA4866) from Pseudomonasaeruginosa PAC1. Proteins 61, 677679

19 Hirano, Y. et al. (2006) Cooperation of multiple chaperones requiredfor the assembly of mammalian 20S proteasomes. Mol. Cell 24,977984

20 Le Tallec, B. et al. (2007) 20S proteasome assembly is orchestrated bytwo of chaperones in yeast distinct pairs and in mammals.Mol. Cell 27,660674

21 Ramos, P.C. et al. (1998) Ump1p is required for proper maturation ofthe 20S proteasome and becomes its substrate upon completion of theassembly. Cell 92, 489499

22 Hirano, Y. et al. (2005) A heterodimeric complex that promotes theassembly of mammalian 20S proteasomes. Nature 437, 13811385

23 Kusmierczyk, A.R. et al. (2008) A multimeric assembly factor controls

Trends in Biochemical Sciences Vol.35 No.11the formation of alternative 20S proteasomes.Nat. Struct Mol. Biol. 15,237244

Review Trends in Biochemical Sciences Vol.35 No.1124 Yashiroda, H. et al. (2008) Crystal structure of a chaperone complexthat contributes to the assembly of yeast 20S proteasomes.Nat. Struct.Mol. Biol. 15, 228236

25 Hirano, Y. et al. (2008) Dissecting beta-ring assembly pathway of themammalian 20S proteasome. EMBO J. 27, 22042213

26 Schmidtke, G. et al. (1997) Maturation of mammalian 20 S proteasome:purification and characterization of 13 S and 16 S proteasomeprecursor complexes. J. Mol. Biol. 268, 95106

27 Marques, A.J. et al. (2007) The C-terminal extension of the beta 7subunit and activator complexes stabilize nascent 20 S proteasomesand promote their maturation. J. Biol. Chem. 282, 3486934876

28 Jager, S. et al. (1999) Proteasome beta-type subunits: unequal roles ofpropeptides in core particle maturation and a hierarchy of active sitefunction. J. Mol. Biol. 291, 9971013

29 Groll, M. et al. (1999) The catalytic sites of 20S proteasomes and theirrole in subunit maturation: a mutational and crystallographic study.Proc. Natl. Acad. Sci. U. S. A. 96, 1097610983

30 Groll, M. et al. (2000) A gated channel into the proteasome core particle.Nat. Struct. Biol. 7, 10621067

31 Whitby, F.G. et al. (2000) Structural basis for the activation of 20Sproteasomes by 11S regulators. Nature 408, 115120

32 Kohler, A. et al. (2001) The axial channel of the proteasome coreparticle is gated by the Rpt2 ATPase and controls both substrateentry and product release. Mol. Cell 7, 11431152

33 Glickman, M.H. et al. (1998) A subcomplex of the proteasomeregulatory particle required for ubiquitin-conjugate degradation andrelated to the COP9-signalosome and eIF3. Cell 94, 615623

34 Rubin, D.M. et al. (1998) Active site mutants in the six regulatoryparticle ATPases reveal multiple roles for ATP in the proteasome.EMBO J. 17, 49094919

35 Lam, Y.A. et al. (2002) A proteasomal ATPase subunit recognizes thepolyubiquitin degradation signal. Nature 416, 763767

36 Husnjak, K. et al. (2008) Proteasome subunit Rpn13 is a novelubiquitin receptor. Nature 453, 481488

37 Schreiner, P. et al. (2008) Ubiquitin docking at the proteasome througha novel pleckstrin-homology domain interaction. Nature 453, 548552

38 van Nocker, S. et al. (1996) The multiubiquitin-chain-binding proteinMcb1 is a component of the 26S proteasome in Saccharomycescerevisiae and plays a nonessential, substrate-specific role in proteinturnover. Mol. Cell Biol. 16, 60206028

39 Jorgensen, J.P. et al. (2006) Adrm1, a putative cell adhesion regulatingprotein, is a novel proteasome-associated factor. J. Mol. Biol. 360,10431052

40 Walz, J. et al. (1998) 26S proteasome structure revealed by three-dimensional electron microscopy. J. Struct. Biol. 121, 1929

41 da Fonseca, P.C. and Morris, E.P. (2008) Structure of the human 26Sproteasome: subunit radial displacements open the gate into theproteolytic core. J. Biol. Chem. 283, 2330523314

42 Forster, A. et al. (2005) The 1.9 A structure of a proteasome-11Sactivator complex and implications for proteasome-PAN/PA700interactions. Mol. Cell 18, 589599

43 Yu, Y.D. et al. (2010) Interactions of PANs C-termini with archaeal 20Sproteasome and implications for the eukaryotic proteasome-ATPaseinteractions. EMBO J. 29, 692702

44 Stadtmueller, B.M. et al. (2010) Structural models for interactionsbetween the 20S proteasome and its PAN/19S activators. J. Biol.Chem. 285, 1317

45 Kohler, A. et al. (2001) The substrate translocation channel of theproteasome. Biochimie 83, 325332

46 Gillette, T.G. et al. (2008) Differential roles of the COOH termini ofAAA subunits of PA700 (19 S regulator) in asymmetric assembly andactivation of the 26 S proteasome. J. Biol. Chem. 283, 3181331822

47 Smith, D.M. et al. (2007) Docking of the proteasomal ATPases carboxyltermini in the 20S proteasomes alpha ring opens the gate for substrateentry. Mol. Cell 27, 731744

48 Saeki, Y. et al. (2009) Multiple proteasome-interacting proteins assistthe assembly of the yeast 19S regulatory particle. Cell 137, 900913

49 Nakamura, Y. et al. (2007) Structure of the oncoprotein gankyrin incomplex with S6 ATPase of the 26S proteasome. Structure 15, 179189

50 Nakamura, Y. et al. (2007) Structural basis for the recognition betweenthe regulatory particles Nas6 and Rpt3 of the yeast 26S proteasome.

Biochem. Biophys. Res. Comm. 359, 50350951 Funakoshi, M. et al. (2009) Multiple assembly chaperones governbiogenesis of the proteasome regulatory particle base. Cell 137, 887899

52 Roelofs, J. et al. (2009) Chaperone-mediated pathway of proteasomeregulatory particle assembly. Nature 459, 861865

53 Le Tallec, B. et al. (2009) Hsm3/S5b participates in the assemblypathway of the 19S regulatory particle of the proteasome. Mol. Cell33, 389399

54 Rabl, J. et al. (2008) Mechanism of gate opening in the 20S proteasomeby the proteasomal ATPases. Mol. Cell 30, 360368

55 Pick, E. et al. (2009) PCI Complexes: Beyond the Proteasome, CSN, andelF3 Troika. Mol. Cell 35, 260264

56 Sharon, M. et al. (2006) Structural organization of the 19S proteasomelid: Insights from MS of intact complexes. PLOS Biol. 4, 13141323

57 Taverner, T. et al. (2008) Subunit architecture of intact proteincomplexes from mass spectrometry and homology modeling. Acc.Chem. Res. 41, 617627

58 Imai, J. et al. (2003) The molecular chaperone Hsp90 plays a role in theassembly and maintenance of the 26S proteasome. EMBO J. 22, 35573567

59 Zuhl, F. et al. (1997) Dissecting the assembly pathway of the 20Sproteasome. FEBS Lett. 418, 189194

60 Leggett, D. et al. (2002) Multiple associated proteins regulateproteasome structure and function. Mol. Cell 10, 495507

61 Ustrell, V. et al. (2002) PA200, a nuclear proteasome activator involvedin DNA repair. EMBO J. 21, 35163525

62 Schmidt,M. et al. (2005) TheHEAT repeat protein Blm10 regulates theyeast proteasome by capping the core particle.Nat. StructMol. Biol. 12,294303

63 Fehlker, M. et al. (2003) Blm3 is part of nascent proteasomes and isinvolved in a late stage of nuclear proteasome assembly. EMBORep. 4,959963

64 Lehmann, A. et al. (2008) Blm10 binds to pre-activated proteasome coreparticles with open gate conformation. EMBO Rep. 9, 12371243

65 Lavabre-Bertrand, T. et al. (2001) Plasma proteasome level is apotential marker in patients with solid tumors and hemopoieticmalignancies. Cancer 92, 24932500

66 Rousseau, E. et al. (2009) Misfolding of proteins with a polyglutamineexpansion is facilitated by proteasomal chaperones. J. Biol. Chem. 284,19171929

67 Borissenko, L. and Groll, M. (2007) Diversity of proteasomal missions:fine tuning of the immune response. Biol. Chem. 388, 947955

68 Zwickl, P. et al. (1999) An archaebacterial ATPase, homologous toATPases in the eukaryotic 26 S proteasome, activates proteinbreakdown by 20 S proteasomes. J. Biol. Chem. 274, 2600826014

69 Benaroudj, N. and Goldberg, A. (2000) PAN, the proteasome-activatingnucleotidase from archaebacteria, is a protein-unfolding molecularchaperone. Nat. Cell Biol. 2 (11), 833839

70 Benaroudj, N. et al. (2003) ATP hydrolysis by the proteasomeregulatory complex PAN serves multiple functions in proteindegradation. Mol. Cell 11, 6978

71 Navon, A. and Goldberg, A.L. (2001) Proteins are unfolded on thesurface of the ATPase ring before transport into the proteasome. Mol.Cell 8 (6), 13391349

72 Zhang, F. et al. (2009) Structural insights into the regulatory particle ofthe proteasome from Methanocaldococcus jannaschii. Mol. Cell 34,473484

73 Zhang, F. et al. (2009) Mechanism of substrate unfolding andtranslocation by the regulatory particle of the proteasome fromMethanocaldococcus jannaschii. Mol. Cell 34, 485496

74 Djuranovic, S. et al. (2009) Structure and activity of the N-terminalsubstrate recognition domains in proteasomal ATPases. Mol. Cell 34,580590

75 Wang, T. et al. (2009) Structural Insights on the Mycobacteriumtuberculosis Proteasomal ATPase Mpa. Structure 17, 13771385

76 Smith, D.M. et al. (2006) Proteasomes and their associated ATPases: adestructive combination. J. Struct. Biol. 156, 7283

77 Smith, D.M. et al. (2005) ATP binding to PAN or the 26S ATPasescauses association with the 20S proteasome, gate opening, andtranslocation of unfolded proteins. Mol. Cell 20, 687698

78 Martin, A. et al. (2008) Pore loops of the AAA+ ClpX machine gripsubstrates to drive translocation and unfolding. Nat. Struct Mol. Biol.

15, 11471151

641

79 Rohrwild, M. et al. (1996) HslV-HslU: A novel ATP-dependent proteasecomplex in Escherichia coli related to the eukaryotic proteasome. Proc.Natl. Acad. Sci. U. S. A. 93, 58085813

80 Bochtler, M. et al. (1997) Crystal structure of heat shock locus V (HslV)from Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 94, 60706074

81 Bochtler, M. et al. (2000) The structures of HsIU and ATP-dependentprotease HsIU-HsIV. Nature 403, 800805

82 Sousa, M.C. et al. (2000) Crystal and solution structures of an HslUVprotease-chaperone complex. Cell 103, 633643

83 Wang, J. et al. (2001) Crystal structures of the HslVU peptidase-ATPase complex reveal an ATP-dependent proteolysis mechanism.Structure (Camb.) 9, 177184

84 Sousa, M. et al. (2002) Crystal structure of HslUV complexed with avinyl sulfone inhibitor: corroboration of a proposed mechanism ofallosteric activation of HslV by HslU. J. Mol. Biol. 318, 779785

85 Park, S.R., J. Kim,W. Robert, J. Schmidt,M. Gygi, SP. Finley, D. (2009)Hexameric assembly of the proteasomal ATPases is templated throughtheir C termini. Nature 459, 866870

Review Trends in Biochemical Sciences Vol.35 No.11642

The 26S proteasome: assembly and function of a destructive machineThe 26S proteasome and its involvement in the ubiquitin-proteasome system (UPS)Prokaryotic CP assemblyEukaryotic CP assembly: -ring formationEukaryotic -ring formation and half-proteasome assemblyOpening doors into the 20S proteasomeOverall structure of the 19S regulatory particleGate opening of the 20S proteasome by the 19S regulatory particleAssembly of the 19S regulatory base complexAssembly of the lid complexConcluding remarks and future perspectivesAcknowledgmentsReferences