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28 Hershko, A. et al. (1994) J. Biol. Chem. 269,

35 Hyver, C. and Leguyader,H. (1990) Biosystems

4940-4946 29 Odell, G. M. (1980) in Mathematical Models in Molecular and Cellular Biology (Segel, L. A., ed.), pp. 647-727, Cambridge University Press 30 Murray, A. W. (1992) Nature 359, 599-604 31 Murray, A. (1994) Curr. Opin. Cell Biol. 6, 872-876 32 Novak, B. and Tyson, J. J. (1995) J. Theor. Biol. 173, 283-305 33 Novak, B. and Tyson, J. J. (1993) J. Cell Sci. 106, 1153-1168 34 Murray, A. W. (1993) Curr. Biol. 3, 291-293

24, 85-90 36 Norel, R. and Agur, Z. (1991) Science 251, 1076-1078 37 Tyson, J. J. (1991) Proc. Natl Acad. Sci. USA 88, 7328-7332 38 Goldbeter,A. (1991) Proc. Natl Acad. Sci. USA 88, 9107-9111 39 Obeyeskere,M. N., Tucker, S. L. and Zimmerman, S. C. (1992) Biochem. Biophys. Res. Commun. 184, 782-789 40 Thron, C. D. (1991) Science 254, 122-123 41 Maddox, J. (1992) Nature 355, 201 42 Dupont, G. and Goldbeter,A. (1992) BioEssays 14, 485-493

PROTEIN DEGRADATION IS now emerging as an important mechanism of cellular regulation. For instance, it is needed for cells to adapt to a changing environment, or in the control of time-dependent cellular programmes. Proteolysis offers some advantages over other possible control mechanisms, as it is fast, therefore enabling the cell to rapidly reduce the level of a defined protein; it is also irreversible, so ensuring complete loss of function. However, the great danger of unspecific degradation of proteins means that proteolysis has to be highly selective. To guarantee this, nature has evolved a remarkably sophisticated proteolytic system: the proteasome. The most common mechanism for marking a protein for degradation via the proteasome is ubiquitination. This involves the attachment of multiple chains of the 76-amino-acid protein ubiquitin to the protein to be degraded. Ubiquitination is performed by a complex enzyme system consisting of E1 (activating) enzymes, E2 (conjugating) enzymes and, in some cases, E3 (ligating) enzymes 1 (Fig. 1). Specificity for the multitude of different substrate proteins is provided by a series of E2 enzymes, which can form heterodimers. Moreover, some E2-dependent ubiquitin conjugation events are aided by substrate-specific E3 enzymes. Several cis-acting signals that indicate a protein should be ubiquitinated have been proposed. One is the 'N-end rule', resulting from the observation that different versions of Escherichia coil 13-galactosidases synthesized in yeast had different proteolytic stabilities, depending on the amino acid present at the amino terminus 2. However, as

W. Hilt and D. H. Wolf are at the Institut for Biochemie,Universit~tStuttgart, Pfaffenwaldring55, 70569 Stuttgart, Germany.

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43 Lauffenburger,D. A. and Linderman,J. J. (1993) Receptors: Models for Binding, Trafficking, and Signaling, OxfordUniversity

Press 44 Perelson, A. S. (1992)in Theory and Control of Dynamical Systems: Applications to Systems in Biology (Andersson, S., Andersson, A. and

Ottoson, U., eds), pp. 200-230, World Scientific Publishing 45 Edgar,B. A., Odell, G. M. and Schubiger,G. (1989) Dev. Genet. 10, 124-142 46 Goldbeter,A. (1995) Proc. R. Soc. London Ser. B 261, 319-324 47 Bray, D., Bourret, R. B. and Simon, M. I. (1993) Mol. Biol. Cell 4, 469-482

Proteasomes: destruction as a programme Wolfgang Hilt and Dieter H. Wolf Proteasomes are large multi-subunit protease complexes that selectively degrade intracellular proteins. Most of the proteins removed by these proteases are tagged for destruction by ubiquitination. Proteasomes have a role to play in controlling cellular processes, such as metabolism and the cell cycle, through signal-mediated proteolysis of key enzymes and regulatory proteins. They also operate in the stress response, by removing abnormal proteins, and in the immune response, by generating antigenic peptides. 26S complex could degrade ubiquitintagged proteins, while the 20S proteasome could not. It became clear that the 20S proteasome functions as the proteolytic core of the larger 26S complex, now called the 26S proteasome. However, whether the 20S proteasome has its own role to play is still an open question. It has been suggested that the 20S and 26S complexes exist in a dynamic equilibrium, and interestingly, the 20S proteasome has been found as a component of larger complexes other than the 26S proteasome 3. The cellular function of these complexes is still unknown. 20S proteason~ have been found in all eukaryotes investigated so far and are located in both the cytoplasm and the nucleus. They are cylindrical partiProteasomes: structure, genes and cles, with a total molecular mass of proteolytic function Several years ago, two large protease approximately 700kDa, and are comcomplexes, which appeared to be differ- posed of numerous low molecular ent entities, were discovered: a multi- weight subunits (molecular masses catalytic protease, or 20S proteasome, 20-35kDa) arranged in a stack of and a larger 26S particle. Subse- four rings, each containing seven quent in vitro studies showed that the subunits 3. yeast cells with a blocked N-end rule pathway do not show an apparent phenotype, this degradation signal does not seem to be involved in vital proteolytic pathways. Other proteins degraded by the proteasome contain 'destruction boxes', which consist of a short stretch of strongly conserved amino acids (Table I). Furthermore, 'PEST' sequences, consisting of regions rich in proline, aspartic acid, glutamic acid, serine and threonine, also seem to act as degradation signals. In fact, deletion of PEST regions in some proteins known to be substrates of the ubiquitin proteasome pathway, caused their partial stabilization (Table I).

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A 20S proteasome has also been found in the archaebacterium Thermoplasma acidophilum, which strongly resembles the eukaro~ic 20S proteasome in its quarternary structure. However, the Thermoplasma proteasome contains only two different types of subunits, known as cr and [3 subunits. Immuno-electron microscopy has demonstrated that the ~ subunits constitute rings at each end of the cylinder, whereas the [3 subunits form the two inner rings, suggesting that the 20S proteasome assembles as an ~7137137(~7 complex4. This has now been confirmed by X-ray crystallographic data for the structure of the Thermoplasma proteasome resolved at a 3.4A resolution 5. The structure shows the 20S proteasome complex to be a hollow cylinder 148]~ in length, 113]~ in diameter Gig. 2) and containing a central channel with three large cavities. The two outer cavities are located at the interfaces between the c~ and [3 rings. The third cavity is located at the centre of the complex and is formed by the 13 rings Gig. 2b,c). This central cavity contains the proteol~ic active sites and access to this compartment is controlled by four narrow gates. The outer gates, which are formed by the ~ subunits, only leave an opening of 13]~ in diameter Gig. 2a). Even though the c~ and [3 subunits show little sequence similarity, the folding of the two subunits is strikingly similar. They exhibit a novel protein topology showing a core sandwich of [3 sheets flanked by (~ helices. The Thermoplasma proteasome is thought to be an ancestor of the eukaryotic 20S proteasome owing to its less complex subunit composition. The sequences of the eukaryotic 20S proteasome subunits (over 60) determined so far, show strong similarities to each other, but lack any detectable similarity to any other lmown eukaryotic proteins. The similarities between these subunits and those from Thermoplasma proteasomes mean that sequences of the eukaryotic 20S proteasome can be divided into two subgroups, ~ and [3 Gig. 3). Further phylogenetic studies subdi~de both the a and 13 subunit groups into seven branches Gig. 3). With the exception of two branches containing the [3 subunits LMP2 and LMP7, which can replace homologous constitutive subunits of the 20S proteasome, only one member from any given organism is found in each branch. This, in

combination with immunoelectron microscopic data 6, suggests that the 20S proteasome is a dimer consisting of two identical subcomplexes, each containing seven different c~ and seven different [3 subunits. 20S proteasomes exhibit at least five distinct proteoUbiquitin l~ic activities against artificial chromogenic peptide substrates 7. Also, they have been shown to degrade insulin A and B chains, and to oxidize proteins in vitro. Screens for yeast mutants with defective peptide-cleaving activities yielded cells bearing mutations in the [3 subunits only, suggesting that the catalytic sites are located within the [3 subunits of the comple~ ,9. This has now been proved for the Thermoplasma proteasome. Mutational studies 1~ as well as X-ray crystallographic 26S proteasome analysis of the binding of a peptide aldehyde inhibitor to the complex 5, identified an amino-terminal threonine of the [3 subunit as the catalytically active amino acid s,l~ Therefore, the proteasome can be considered Figure 1 as a novel type of protease, The ubiquitin-proteasome degradation pathway. which can be added to the Ubiquitin is activated by forming a thioester bond with serine-, cysteine-, asparticthe ubiquitin-activating enzyme E1 using its carboxyl and metallo proteases. terminus. Ubiquitin is then transferred to the active The X-ray data also cysteine group of a ubiquitin-conjugating enzyme E2. show that the 14 proteoE2 enzymes attach ubiquitin to the eamino groups of lysine residues of substrate proteins. This process, l~ically active sites of the in some cases, needs cooperation with a ubiquitin ligThermoplasma proteasome ase E3. Repeated conjugation of ubiquitin to lysine are located inside the cenresidues of formerly bound ubiquitin moieties leads tral cavity of the complex to the formation of multi-ubiquitin chains. Multi(Fig. 2c). Degradation studubiquitinated substrate proteins are recognized by ies with Thermoplasma prothe 26S proteasome and degraded. Muiti-ubiquitin chains are released from the complex and ubiquitin teasomes in vitro have is recycled. demonstrated that a protein must be unfolded in order to be degraded by this complex u. Electron the inner active compartment of the microscopic studies demonstrated that complex. 20]k gold particles bound to unfolded 26S proteasomes. By contrast to 20S insulin B chains are attached specifi- proteasomes - which in vitro will only cally to the top and bottom of the degrade certain denatured or oxidized Thermoplasma proteasome cylinder, proteins in the absence of ATP - 26S probut cannot pass through the narrow teasomes degrade ubiquitin conjugates gate u that controls access to the inner in an ATP-dependent reaction. 26S compartment s. These data fully support proteasomes are large protease comthe mechanistic model put forward pre- plexes with a molecular mass of appro~viously~2, in which proteolysis by the mately 17001d)a and contain the 20S proteasome requires unfolding and proteasome as a functional core 3. transport of the substrate protein to Electron microscopic images of the 26S j

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Table I. Substrates of the 26S proteasome a

Cellular function

Degradation Mediator/E2 enzyme signal Notes

Metabolic enzyme; polyamine synthesis

Antizyme

?

Metabolic enzyme; gluconeogenesis

Ubcl, Ubc 4, Ubc 5

?

Transcriptional activator; amino acid and purine synthesis

Ubc2, cdc34 ubc3

Catabolite inactivation, 39 glucose induced PESTDecreased upon 40 sequences starvation

Transcriptional repressor; repression of mating-type specific genes G protein; signal transduction

Ubc4, Ubc5, Ubc6, Ubc7 Ubiquitin

Degradation box N-end rule

G1 cyclin; control of cdc28 kinase

cdc34 ubc3

Cin3

G1 cyclin; control of cdc28 kinase

cdc34 ubc3

Clb5

S-phase cyclin; control of cdc28

Ubc9

Clb2

Mitotic cyclin; control of cdc28 kinase

Ubc9

Sic1

CDK inhibitor; inhibition of CIb5-cdc28 complex Protein kinase; meiotic arrest in oocytes

cdc34 ubc3

Transcriptional activator; signal transduction Tumour suppressor; cell cycle pause

Ubiquitin

PESTsequences PESTsequences Destruction box Destruction cdc16/cdc23box dependent Control of DNA replication AminoOncogenic when terminal stabilized 2nd amino acid ~:lomain Oncogenic when stabilized Oncogenic if destabilized, E6-E6AP-mediated

Transcriptional regulator; immune and inflammatory response Inhibitor of NF-KB; immune and inflammatory response

Ubiquitin

?

Processing

?

?

Complete degradation 32

Substrate protein Metabolic adaptation Ornithine decarboxylase Fructose-%6biphosphatase Gcn4

Cell differentiation MAT (~2 repressor

G(~ Cell-cycle control/ cell growth CIn2

c-Mos

c-Jun p53

Stress response NF-KB I-KB

Removal of waste Canavanyl proteins Fas2

CTFR

Ubiquitin

Ubc4h

Undefined

Ubcl, Ubc4, Ubc5

c~-subunit of fatty acid synthase

?

Ion pump; chloride transport across plasma ? membrane

Refs

Ubiquitin independent 37, 38

9, 41 42

22 23 25 25, 26, 3O 27 43, 44

45 46

32

Abnormal, contain 8, 9 arginine analogue Degraded when not 9 assembled with Fasl Mutant responsible 47, 48 for cystic fibrosis

?

aAbbreviationsused: CDK,cyclin-dependentprotein kinase; CTFR,cystic fibrosis transmembraneconductanceregulator.

proteasome show the typical 20S proteasome structure with two additional substructures attached at both ends. These 19S cap complexes are composed of at least 15 different subunits with molecular masses ranging from 25kDa to 110kDa that associate with the 20S proteasome in an ATP-dependent

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manner. It is thought that the 19S cap complexes are required for the recognition of ubiquitinated proteins, as well as to unfold and transport substrate proteins to the proteolytic active 20S core. ATPase subunits of the 19S cap. A set of 19S cap subunits have been characterized as members of a novel family of

ATPases, now called AAA-ATPases13. Interestingly, some of these I9S cap proteins are similar or identical to proteins that are involved in transcriptional activation. For example, the human $4 subunit has strong similarities to the TATA-box-binding protein-1 (TBP1) 14, which interacts with the

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human immunodeficiency virus (HIV) transcriptional activator protein TAT in vitro. Subunit $7 is identical to MSS1, a modulator of HIV TAT-dependent transcriptional activation 14, and the human $6 subunit, previously described as TBP7, is a homologue of TBP1 ~ef. 14). Two yeast genes CIM3 and CIM5 have been cloned whose protein products also show strong similarity to human 19S cap subunits is. Cim5 exhibits 70% similarity with human S7/MSS1 and can be functionally replaced by the human protein. CIM3 is identical to a formerly cloned yeast gene, SUGI ~ef. 16). Further evidence that Cim3 and Cim5 are components of the 26S proteasome has been obtained from mutations in the genes encoding these proteins that lead to a defect in ubiquitin-dependent proteolysis. Interestingly, genetic studies showed that SUGI/CIM3 also functions in transcriptional activation. Moreover, Sugl is a component of the RNA polymerase II complex 17. In Schizosaccharomyces pombe, a 19S cap subunit encoded by the mts2 § gene has been identified 18. The protein encoded by the mts2 § gene has 75% identity with the human $4 subunit, which can also functionally replace it. Polymerase chain reaction ~CR) using oligonucleotide primers derived from highly conserved boxes within regions characteristic for the AJ~A,-ATPaseshas yielded 11 members of this ATPase family in yeast. These members have been named YTA1-YTAll ~ef. 19). In addition to YTA3 (which is identical to CIM5), YTAI, YTA2 and YTA5 are proposed to code for additional yeast 19S cap ATPase components, based on sequence similarity. ATPase subunits of the 26S proteasome are thought to be responsible for the unfolding and the transport of substrate proteins into the 20S core. However, the detailed mechanism has yet to be discovered. Non-ATPase subunits. Several more 19S cap subunits have been identified, which do not belong to the ATPase family. The human $5 subunit was found to bind multi-ubiquitin chains and, therefore, seems to be responsible for recognition of the ubiquitin-protein conjugates 14. The genes encoding four additional non-ATPase subunits of the human 19S cap have been cloned 2~ The largest subunit of the 19S subcomplex, p112 ($1), is homologous to a yeast protein encoded by the SEN3 gene, which has been shown previously to influence the

tRNA-splicing endonuclease system. Subunit p31 was uncovered as a homoiogue of the yeast Ninl protein, which is involved in cellcycle control, and the human p28 and p58 subunits have been identified as homologues of yeast proteins encoded by the SUNI and SUN2 genes, respectively. Both of these yeast genes are multicopy suppressors of the temperature sensitivity of a ninl-I mutant. The yeast gene DOA4 originally found by screening for mutants with a defect in degradation of the MATa2 repressor codes for a deubiquitinating enzyme responsible for release of ubiquitin from the substrate protein. As a de-ubiquitinating activity is linked to the 26S proteasome, so Doa4 has been linked with this complex21.

(a)

Cellular functions of the 26S proteasome Proteasomes are essential to life. Studies of yeast knockout mutants revealed that proteasomes fulfil vital functions in the cell. With one exception, individual chromosomal deletions of each of the 14 known yeast 20S proteasome genes are lethal 8,9, as is chromosomal Figure 2 deletion of the 19S cap Three dimensional X-ray structure of the Thermogenes CIM3, CIM5 (Ref. 15), plasma proteasome. (a} Top view of the 20S YTAI or YTA2 (Ref. 19). proteasome (Ca atoms only), showing the 13/~ A variety of substrates of entrance to the channel. (b) View of the proteasome cut open along the sevenfold axis. Reproduced with the 26S proteasome have permission from Ref. 5. (r Schematic drawing of a been characterized, allowing proteasome cut open showing the three cavities, the insight into the different gates and the proteolyticallyactive sites. cellular functions of the enzyme complex. These include: regulation of metabolic adaptation, cell differentiation, specific cyclins. There is evidence that cell-cycle control, a role in the stress proteasomes might be important for response and removal of abnormal pro- cell-cycle regulation by degrading teins (Table i). cyclins that act in different phases of the cell-cycle. In Saccharomyces Cell-cycle control cerevisiae, cyclins Clnl and Cln2 are The eukaryotic cell-division cycle is synthesized in late G1 phase and are controlled by cyclin-dependent protein crucial for progression from G1 to kinases (CDKs). The appearance and 'Start' of a new cell cycle. In the ensuing disappearance of particular active ki- S phase, both proteins disappear as a nase complexes during different phases result of proteol~ic degradation. In of the cell cycle is regulated by synthe- contrast, levels of Cln3, which is also sis and proteol~ic degradation of important for progression from G1 to

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r~ hs-C9

I'm-c9

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L x1-C9-1 dm-PROS29 dd-4 sc-Y13 . m-C3

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._L-•• 13

14

Figure 3 Dendrogram showing the relationships among known eukaryotic 20S proteasome protein sequences. The dendrogram was created by the CLUSTAL multiple sequence alignment program using the DNASTAR'~ 'Lasergene' software. The 14 main branches are numbered. Abbreviations for species are: at, Arabidopsis thaliana; dd, Dictyostelium discoideum; dm, Drosophila melanogaster, gg, Gallus gallus; hs, Homo sapiens; mm, Mus musculus; rn, Rattus norvegicus; sc, Saccharomyces cerevisiae; sp, Schizosaccharomyces pombe; xl, Xenopus

laevis.

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Start, do not change throughout the cell cycle, despite Cln3 also being susceptible to proteolytic degradation. Proteolysis of the G1 cyclins Clnl, Cln2 and Cln3 depends on the presence of PEST sequences located at the carboxyl terminus. Cln2 has been shown to be multi-ubiquitinated by the E2 enzyme cdc34ubc3 suggesting that proteasomes are responsible for its degradation 22. This has also been demonstrated for Cln3 degradation 23. Genetic studies in yeast have also provided clues regarding the function of proteasomes in S, G and M phases. The ninl-I mutants, which contain a mutation in a non-ATPase regulatory subunit of the 26S proteasome, are blocked in both the GI-S transition and the G2-M transition at restrictive temperatures. Yeast mutants cim3-1 and cim5-1 harbouring mutations in the ATPase subunits of the 26S proteasome exhibit a temperature-sensitive cell-cycle phenotype ~s. At the non-permissive temperature, these mutants stop growth with replicated DNA and short intranuclear spindles; characteristics exhibited by S. cerevisiae cells arrested in G2-metaphase. A similar phenotype has been observed in 26S proteasome mutants (mts2) of S. pombe 18. Deficiency in the degradation of ubiquitinated proteins by the cim3, cim5 and mts2 mutants suggests that such cell-cycle phenotypes are a result of defective proteolysis ~sJS. In fact, B-type cyclins, which are needed during S, G2 and M phases, are also degraded by the ubiquitin-proteasome pathway: studies on the rapid mitotic degradation of B-type cyclins revealed ubiquitin modification before proteolysis 24. By contrast to the PEST-sequencedependent proteolytic instability of G1 cyclins, rapid degradation of B-type cyclins is signalled by destruction boxes. Cyclin Clb5, which acts during S-G2 phase, is degraded in a Ubc9dependent fashion. Moreover, in contrast to wild-type cells, overexpression of CLB5 is not tolerated by mutant cells defective in the Prel subunit of the 20S proteasome 2s. A similar result has been obtained for the mitotic cyclin Clb2. Overexpression of CLB2 caused prel-I mutant cells to stop growth 26. Inhibitors of CDKs are also controlled by proteolytic destruction: whereas cyclin-degradation is needed to shut off active CDKs, proteolysis of CDK inhibitors results in turning on CDK activity. The S. cerevisiae Sic1 protein is a CDK inhibitor that interacts with the

MARCH 1 9 9 6

Clb5-cdc28 kinase complex (cdc28 is the yeast homologue of the cdc2 kinase) causing cell-cycle arrest before DNA replication. To activate the Clb5-cdc28 kinase complex and to restart DNA replication, Sic1 has to be degraded. Genetic evidence showed that Sic1 is ubiquitinated by the ubiquitin-conjugating enzyme cdc34, suggesting its degradation by the proteasome 27. Recent work has demonstrated that destruction of other proteins most probably degraded by the proteasome, might be a critical step in the cell cycle. For example, in mammalian cells, a large anaphase-promoting complex (APC) containing CDC16 and CDC27 components has been discovered. This complex was shown to be an E3 enzyme, which in cooperation with Ubc4 mediates degradation of B-type cyclins28. Immunological studies located CDC27Hs and CDC16Hs to the centromere and the mitotic spindle 29. In yeast, cdcl6 and cdc23 mutants (cdc23 is also a component of the APC complex) are defective in Clb2 degradation. Previous work demonstrated that degradation of Cib2 is required for exit from mitosis. However, cdcl6 and cdc23 mutants arrest before anaphase, with duplicated DNA and short mitotic spindles. Thus, besides controlling Clb2 destruction during mitosis, CDC23 and CDC16 are required at the metaphaseanaphase transition. It became clear that cdc23 mutants cannot enter anaphase because they fail to separate sister chromatids. This finding, together with additional genetic data, led to the suggestion that the cdc23-containing complex mediates proteolytic destruction of a so far unknown protein responsible for holding sister chromatids together 3~

Stress responseand the immunesystem Yeast mutants with defects in the different activities of the 20S proteasome are sensitive to heat stress and to the arginine analogue canavanine, which induces formation of abnormal proteins. Under such stress conditions these mutants accumulate ubiquitinated proteins &9. In mammalian cells, the activity of the 20S proteasome can be blocked by specific inhibitors, which also leads to stabilization of abnormal proteins induced with canavanine3L NF4cBis a transcriptional regulator of a multitude of genes involved in the immune and inflammatory responses. These genes encode inflammatory and

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chemotactic cytokines, hematopoietic growth factors, cell adhesion molecules, antibodies, class I MHC molecules and cytokine receptors. NF-KBis highly regulated and is activated by a great variety of mostly pathogenic stimuli, such as viruses, bacteria, energyrich radiation, oxidants and the inflammatory cytokines TNF-cr and IL-113.In its active form NF-KB is a heterodimeric complex consisting of a p50 subunit (NF-KB1) and a p65 subunit (RelA). NF-KB is kept inactive by two different mechanisms: (1) association of the inhibitor protein I-KBc~to the p50-p65 complex; and (2) synthesis of NF-KB1 as a 105kDa precursor protein that can assemble with the p65 subunit, but forms an inactive complex. Recent studies demonstrated that proteasomes are responsible for activation of NF-KB by degradation of I-KBa as well as by processing of the 105kDa NF-KB precursor to the active p50 form32. This has been shown by proteasome depletion and inhibition studies in cell extracts, and in vivo, using yeast 20S proteasome mutants that are defective in proteolysis. Interestingly, these results demonstrate that proteasomes are not only responsible for complete degradation of proteins, but are also required for activation of proteins by processing of inactive precursors.

Antigen presentation Most vertebrate cells present short fragments of intracellular proteins on the cell surface, which initiate the immune response. Strong evidence now suggests that such antigenic peptides are generated by proteasome-dependent degradation. Studies with cells containing a thermosensitive defect of ubiquitin conjugation demonstrated that antigen presentation needs an active ubiquitin system 33. Two genes, LMP2 and LMP7, coding for non-essential 13-type subunits of the 20S proteasome, are located within the class II MHC region adjacent to the genes TAP1 and TAP2. These genes encode proteins that shuttle antigenic peptides from the cytoplasm to the endoplasmic reticulum, where they bind class I MHC complexes, and are then transported to the cell surface. Expression of LMP2 and LMP7 is induced by ~-interferon, whereas expression of the genes encoding the homologous housekeeping 20S proteasome subunits Y (hs-delta) and X is suppressed 2~ The induced protein products of LMP2 and LMP7 replace housekeeping 20S proteasome subunits,

which alters the peptidecleaving activities of the complex without affecting the ubiquitin-conjugate-degrading activity of the 26S proteasome 34. Recently, a third human 20S proteasome subunit, MECL1, has been shown to replace subunit Z upon induction with ~/-interferon2~ Evidence for the function of proteasomes in presenting antigens also came from proteasome inhibition studies in lymphoblast cells, in which proteasome inhibitors blocked the generation of peptides presented by the class I MHC molecules3L Moreover, LMP7-defective mice show reduced levels of class ! MHC cell-surface expression and inefficient antigen presentation 35. Owing to their altered peptide cleaving activities, Lmp2- and Lmp7-containing 'immunoproteasomes' are suggested to improve antigen presentation by favouring the production of peptides appropriate for binding to class I MHC molecules36.

Concludingremarks The proteasome may be considered to be a functionally sophisticated counterpart of the ribosome. Regulation of protein levels by destruction via the proteasome is an essential cellular tool. It is obvious that the few substrates of proteasomes discovered to date represent only the tip of the iceberg and it will be a great challenge to uncover all the cellular processes that proteasomes are involved in, as well as the detailed mechanisms underlying these selective processes.

Acknowledgements We thank R. Huber, J. L6we, W. Baumeister, E. Seemiiller, K. Tanaka, A. L. Goldberg, K. Hendil, K. Ferrel, M. Rechsteiner, S. Wilk and B. Dahlmann for providing manuscripts of their work prior to publication. Thanks to J. LOwe, D. Stock and R. Huber also for providing Fig. 2a,b. The work of the authors was supported by the Deutsche Forschungsgemeinschaft (Bonn), the German Israeli Foundation for Scientific Research and Development (Jerusalem) and the Fonds der Chemischen Industrie (Frankfurt). We would also like to acknowledge all those whose work has not been cited owing to a limit in the number of references permitted.

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IN EUKARYOTES, the entire genome is replicated precisely once during each S phase of the cell cycle. Failure to replicate even small sections of DNA can have disastrous consequences when the sister chromatids are pulled apart during mitosis. Over-replication of DNA is also likely to be harmful for the cell, as it would represent an irreversible genetic change, potentially leading to gene dosage problems and the risk of recombination occurring in the duplicated region. To ensure the precise replication of eukaryotic chromosomes, many thousands of replication origins must each fire once and only once in each cell cycle. A model based on observations in frog eggs requires the action of two distinct signals for initiation at any given replication origin (Fig. 1). The first signal, replication licensing factor (RLF), 'licenses' replication origins by putting them into an initiation-competent state (Fig. lb). The second signal, S-phasepromoting factor (SPF), induces licensed origins to initiate, and in doing so removes the license (Fig. lb). As long as the licensing signal and the initiation signal act sequentially, and never act on DNA at the same time, the result will be the precise duplication of the DNA. If the two signals did act on DNA at the same time, this would lead to repetitive cycles of licensing and initiation with consequent re-replication of sections of the DNA. Licensing and initiation signals have been analysed in cell-free extracts of Xenopus eggs that support chromosomal DNA replication in vitro. In this system, RLF (the license) and SPF (the initiation signal) are prevented from acting on DNA at the same time in two different ways. First, RLF cannot cross the nuclear envelope and so it can only license DNA when the nuclear envelope J. P. J. Chong, P. Th6mmes and J. J. Blow are at the ICRF Clare Hall Laboratories, Blanche Lane, South Mimms, Potters Bar, Herts, UK EN6 3LD.

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The role of MCM/PI proteins in the licensing of DNA replication James P. J. Chong, Pia Th6mmes and J. Julian Blow The DNA replication licensing system ensures that eukaryotic chromosomes replicate precisely once per cell cycle. A central component of the licensing system, RLF-M, has recently been shown to consist of a complex of Mcm/P1 proteins. This result allows us to integrate data about the MCM/P1 family obtained in different eukaryotes, ranging from yeast to man, into a general picture of the way that chromosome replication is controlled. has broken down in mitosis 1. By con- which are required for licensings. RLF-M trast, SPF can only initiate DNA repli- has been purified to apparent homocation on licensed DNA within an intact geneity and shown to consist of a comnucleus. Thus, active SPF and active plex of at least three polypeptides, with RLF can never act on the DNA at the molecular weights of 92kDa, 106kDA same time. Second, the spatial separ- and 115 kDa. By immunological analysis ation provided by the nuclear envelope it was shown that the 106kDa polypepis reinforced by a temporal separation, tide is Xenopus Mcm3, while the other as both activities are periodic in the cell two polypeptides are also members cycle. RLF is activated abruptly during of the Mcm (minichromosome mainthe metaphase-anaphase transition and tenance) family, probably homoiogues decays during interphase 2 (Fig. lc); SPF of Mcm5 and Mcm2 (Ref. 5). Immunodepletion of the RLF-M comactivity, in contrast, can only be detected during interphase 3 (Fig. ld). The plex using anti-Mcm3 antibodies abolexistence of two distinct mechanisms ished RLF-M activitys,6. The overall that separate the licensing and initi- behaviour of the purified RLF-M comation signals emphasizes the crucial ponent is similar to the license shown importance of this control system to in Fig. lb in that: (1) RLF-M, in conjunction with RLF-B, is required for each the cell. successive round of DNA replicationS; (2) Mcm3 associates with chromatin in Mcm proteinsin the RLF-Mcomplex RLF has recently been subject to the presence of RLF-B, but is removed biochemical analysis, using an assay during replicationS; and (3) reassocisystem based on the ability of certain ation of Mcm3 with chromatin, necesprotein kinase inhibitors to inhibit RLF sary for a further round of replication, activation at the metaphase-anaphase requires the permeabilization or breaktransition 2,4. RLF activity in Xenopus can down of the nuclear envelopes,s. The be separated into two essential com- licensing system is likely to be similar ponents, RLF-M and RLF-B, both of in mammalian cells, as Xenopus extracts 9 1996, Elsevier Science Ltd

PII: S0968-0004(96) 10013-X