doi:10.1006/jmbi.2001.5063 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 313, 465±471

COMMUNICATION

Reconstitution of Hybrid Proteasomes from PurifiedPA700-20 S Complexes and PA28aaabbb Activator:Ultrastructure and Peptidase Activities

Friedrich Kopp1, Burkhardt Dahlmann2 and Lothar Kuehn1*

1Department of ClinicalBiochemistry, DeutschesDiabetes-ForschungsinstitutDuÈ sseldorf, Germany2Institute of Biochemistry/ChariteÂ, Humboldt-UniversitaÈtBerlin, Berlin, Germany

complex, preventing access of p

E-mail address of the correspondlothar.kuehn@dd®.uni-duesseldorf.d

Abbreviations used: MHC, majorcomplex; MCA, 7-amino-4-methylcobenzoyl; Z, benzoyloxycarbonyl; Suendoplasmic reticulum.

0022-2836/01/030465±7 $35.00/0

The activity of the proteasome, the major non-lysosomal proteinase ineukaryotes, is stimulated by two activator complexes, PA700 and PA28.PA700-20 S-PA700 proteasome complexes, generally designated as 26 Sproteasomes, degrade proteins, whereas complexes of the type PA28-20 S-PA28 degrade only peptides. We report, for the ®rst time, the in vitroreconstitution of previously identi®ed hybrid proteasomes (PA700-20S-PA28) from puri®ed PA700-20 S proteasome complexes and PA28activator. In electron micrographs, the hybrid appears as a corkscrew-shaped particle with a PA700 and a PA28 activator each bound to aterminal a-disk of the 20 S core proteasome. The multiple peptidaseactivities of hybrid proteasomes are not different from those of PA28-20 S-PA28 or PA700-20 S-PA700 complexes.

# 2001 Academic Press

Keywords: human; proteasome; activator; hybrid; electron microscopy

*Corresponding author

The 20 S proteasome or multicatalytic proteinaseis a highly conserved, multisubunit complex. Ineukaryotes, it is a central component of the majornon-lysosomal proteolytic pathway, present in thenuclear and cytosolic compartments.1 The cylindri-cal particle of about 700,000 Mr comprises 28subunits, arranged in a stack of four heptamericrings aligned in the order abba. The seven structu-rally similar a and b subunits occupy de®nedpositions within the respective ring, resulting in ana1-7b1-7b1-7a1-7 complex.2 ± 6 The 20 S proteasomecan thus be regarded as a complex dimer.2

Eukaryotic proteasomes display distinct trypsin-like, chymotrypsin-like and post-glutamylpeptidehydrolyzing activity. These reside in separateb-subunits, each of which is replaced by g-interfer-on-inducible subunits to form theimmunoproteasome.7 X-ray crystallography ofyeast proteasomes has shown that the N termini of®ve of the a-type subunits extend into the centre ofthe a-ring and block the opening at each end of the

rotein substrates to

ing author:e

histocompatibilityumarylamide; Bz,c, succinyl; ER,

the innermost chamber which harbours the cataly-ticb-subunits.5 Thus, interaction between 20 S coreparticles and regulatory complexes like PA700(19 S regulator complex) or PA28 (11 S activator)probably opens the proteasome channel for entryof the protein substrate.8 In fact, a recent analysisof co-crystals of PA26 activator (PA28 eqivalent)from Trypanosoma brucei and yeast 20 S proteasomehas shown that binding of the activator to theterminal a-disk displaces these blocking peptidesegments and opens the axial gate.9 Of these twoprotein factors which stimulate 20 S proteasomalactivity, PA700 is a large, 700 kDa ATPase-containing regulatory complex which, in thepresence of ATP, binds to the a-rings of the 20 Sproteasome to form an even larger complex whichcatalyses the ATP-dependent degradation of ubi-quitinylated proteins.10,11 This PA700-20 S-PA700complex, generally designated 26 S proteasome,has a molecular mass of 2.02 � 106 and a sedimen-tation coef®cient of 30.3 S.12 PA28, the otherstimulatory protein, is found in species with animmunocompetent system.13 PA28 is composed ofPA28a and PA28b, two different but homologoussubunits which form the active heteromultimerwith a molecular weight of about 200,000. The twosubunits are assembled into a heterohexamer(a3b3)14 ± 16 or a heteroheptamer (a3b4).

17 PA28 acti-

# 2001 Academic Press

Figure 1. Separation of free 20 S, single and double-capped PA700-20 S proteasome complexes by glyceroldensity gradient centrifugation. (a) Chymotrypticactivity pro®le showing the fractionation of 26 S protea-some complex on a 20 %-40 % (v/v) glycerol gradient inlow ionic strength buffer (see below). (b) Electronmicro-graphs of protein from peak one, two and three, follow-ing glycerol gradient centrifugation and fractionation asin (a). Peak one, banding at about 29 % glycerol, showsfree 20 S particles, almost exclusively in end-on views;peak two and three proteins, banding at about 31 % and32 % glycerol respectively, show the single and double-

466 Hybrid Proteasomes of Human Blood Cells

vates 20 S proteasomal peptidase activities, but notthe proteinase activity13 by binding to the a ringsof the proteasome without requirement of ATP.18

PA28 has been implicated in the major histocom-patibility complex (MHC) class I antigen proces-sing pathway as it favours the production ofdominant class I ligands in vivo19 and in vitro20 andan impaired immune response has been reportedfor PA28 knockout mice.21

As the PA28-20 S-PA28 complex cannot digestintact native or ubiquitinylated proteins, it mustact either downstream to the PA700-20 S-PA700proteasome which degrades multiubiquitin-labeledproteins, or a PA28-20 S-PA700 proteasome exists

capped PA700-20 S complex. Magni®cation is 150,000times. (c) Speci®c chymotryptic activity of 2 nM protea-some complex protein from peak one, two and three,respectively in the absence (open columns) and presence(shaded columns) of equimolar amounts of proteasomeactivator PA28. Values given are means � SEM fromfour separate experiments. Methods: For negative stain-ing, glow-discharge-activated carbon-coated Formvargrids were used which had been pretreated for 15 min-utes with 2 % (w/v) uranyl acetate prior to adsorptionof proteins and followed by a one minute staining with2 % uranyl acetate. PA28 activator protein was puri®edto homogeneity from outdated human blood exactly asdescribed for rabbit erythrocytes.22 Prior to electronmicroscopy, the sample buffer was exchanged for a buf-fer consisting of 20 mM TRIS-HCl, 1.1 mM MgCl2,0.1 mM EDTA, 1 mM DTT, 1 mM NaN3, 2 mM ATP,30 % (v/v) glycerol pH 7.5 (low ionic strength buffer).Buffer exchange was performed by size-exclusion chro-matography on a Superdex 200 PC 3.2/30 column inconjunction with the Amersham-Pharmacia SMART2

chromatography system. PA28, eluting between 1.25and 1.35 ml was used in all subsequent experiments.Puri®cation of 26 S proteasomes from human blood wasessentially as described by Hough et al.28 as modi®ed byDahlmann et al.29 with buffers containing 10 % (v/v)glycerol and 2 mM ATP throughout. The ®nal prep-aration, obtained after Arginine-Sepharose chromatog-raphy and comprising free 20 S, 20 S-PA700 proteasomeand PA700-20 S-PA700 proteasome complexes, was con-centrated by centrifugation for 24 hours at 35,000 rpmin a Beckman Ti45 rotor. A 120 mg sample of the concen-trated protein sample was loaded onto a 20 %-40 % (v/v) glycerol gradient in 20 mM TRIS-HCl, 1.1 mMMgCl2, 0.1 mM EDTA, 1 mM DTT, 1 mM NaN3, 2 mMATP, pH 7.5 (low ionic strength buffer). After 24 hoursat 25,000 rpm in a Beckman SW40 Rotor and fraction-ation (0.4 ml/fraction), 10 ml aliquots were assayed forchymotryptic activity, with Suc-LLVY-MCA at 100 mM®nal concentration. After 15 minutes at 37 �C, the reac-tion was stopped and the reporter group was monitoredspectro¯uorimetrically as described.22 The effect ofPA28ab activator protein on speci®c chymotrypticactivity of peak one, two and three proteasome com-plexes was determined by adding, to each peak fraction,an about equimolar amount of PA28ab. Preincubationlasted for 15 minutes at room temperature. Further

assay conditions were as described above.

Figure 2. Electronmicrographs of (a) PA28 activator and (b) 20 S-PA700 proteasome complex used for reconstitu-tion of PA28-20 S-PA700 hybrid proteasomes. (a) PA28 activator complex (2.5 mg/ml) displays a cup-shaped struc-ture, appearing either as circular-shaped 10 nm objects in end view or as structures with U-shaped contours in sideview as indicated by arrows in the inset (c). (b) This ®eld shows proteins banding at 30.8(�0.4) % glycerol (n � 6), fol-lowing glycerol gradient centrifugation and comprising the puri®ed 20 S-PA700 proteasome complex from peak twoin Figure 1, at a protein concentration of 30 mg/ml. Magni®cation is given by bars as indicated in (a)-(c). Methods:Electron microscopy and puri®cation of PA28 activator as well as PA700-20 S single-capped proteasome complexwere as described in the legend to Figure 1.

Hybrid Proteasomes of Human Blood Cells 467

that performs the two-step proteolytic processwithin the same complex, degradation to largepeptides and ®nal trimming.

The existence of such a complex has beensuggested by our earlier experiments, showing thatfor the formation of the PA28-20 S complex frompuri®ed PA28 and 20 S proteasome, peptidaseactivity is almost fully effected by the binding of asingle PA28 activator,22 leaving the second a-diskof the 20 S proteasome available for binding of afurther regulator molecule. Similar results havebeen reported for the formation of proteasomecomplexes from PA700 and 20 S, where binding ofa single PA700 activator elicited enhanced pepti-dase activity.23

Subsequent studies by Hendil et al.24 as well asby Tanahashi et al.25 have demonstrated, by use ofspeci®c antibodies, simultaneous binding of PA28and PA700 to 20 S proteasome, and have identi®edit as a PA28-20 S-PA700 complex, termed hybridproteasomes.25 Thus far, attempts at biochemicalisolation of hybrid proteasomes to conduct func-tional studies have been unsuccessful. We thereforetook an alternative approach to reconstitute such

hybrid complex from isolated 20 S-PA700 andPA28 proteins.

As viewed in the electron microscope, samplesof biochemically puri®ed and apparently homo-geneous ``26 S'' complex generally consist of a mix-ture of PA700-20 S and PA700-20 S-PA700complexes as well as free 20 S particles (and freePA700) in varying proportion, depending on suchfactors as tissue origin, method of isolation andage. It is clear that for optimal yield of hybrid pro-teasomes by in vitro reconstitution and a functionalcomparison of the different proteasome complexes,a highly puri®ed sample of PA700-20 S is requiredas reaction partner. Toward this end, we subjectedthe 26 S proteasome sample, obtained with ourpuri®cation protocol, to an additional glycerolgradient centrifugation. Among the various par-ameters tested, protein load, ionic strength of thebuffer, as well as the concentration-range of theglycerol gradient were found to be crucial for effec-tive resolution and to minimize dissociation of therelatively labile single and double-capped PA700-20 S complexes. As illustrated in Figure 1(a), witha moderate protein load, 26 S proteasomes sub-

468 Hybrid Proteasomes of Human Blood Cells

jected to centrifugation on a 20 %-40 % glycerolgradient in a low ionic strength buffer are resolvedinto three peaks of peptide-hydrolyzing activity.According to electron miscroscopic analysis(Figure 1(b)), these correspond to free 20 S protea-somes (peak one) consistently banding at about29 % (28.7(�0.3), n � 6) glycerol, with peak twoand peak three protein corresponding to the singleand double-capped PA700-20 S complex, bandingat about 31 % (30.8(�0.4), n � 6) and at about 32 %(32.4(�0.3), n � 6) glycerol, respectively. Free 20 Sparticles occasionally seen on the micrographs ofpeak two and three proteins and single-cappedPA700-20 S complexes in the latter re¯ect partialdissocation of these complexes, possibly promotedby the staining procedure which is performed atacidic pH and high ionic strength.

Determination of speci®c chymotryptic activity(Figure 1(c)) reveals that equimolar amounts of20 S, PA700-20 S and PA700-20 S-PA700, hydro-lyze the substrate at a rate of 150, 600 and2030 pmol/ml per minute, respectively. Thus, theactivity of 20 S proteasomes is stimulated onlyabout fourfold upon binding of one PA700 regula-tor to form the 20 S-PA700 proteasome complex, asopposed to an about 11-fold stimulation of thehydrolysis rate attained with the double-cappedcomplex. Upon addition of equimolar amounts ofpuri®ed PA28 activator, hydrolysis rates areincreased with peak one and two (with no furtherstimulation seen when higher amounts of PA28 areadded), attaining values indistinguishable fromthat seen with PA700-20 S-PA700 complex of peakthree, whose activity remains unchanged in thepresence of PA28. This result clearly shows thatcapping of the 20 S proteasome by a single PA700activator molecule leads to submaximal activityonly, and that maximal activation can be achievedby binding of a PA28 activator to the free, seconda-disk of the 20 S-PA700 complex. Moreover, PA28activator has no stimulatory effect on peak threeactivity. If this complex were substantially con-taminated with the single-capped particle, free 20 Sor both, basal activity should be lower and addedPA28 be expected to have a further stimulatoryeffect. We therefore conclude that this fractioncomprises primarily double-capped PA700-20 Scomplexes. Furthermore, a count of particles onelectronmicrographs of peak one protein showsthat 99 % correspond to free 20 S proteasomes.Finally, as peak two and three proteins are devoidof free 20 S proteasomes when analyzed by nativegel electrophoresis (results not shown), we inferthat peak two protein consists primarily of thesingle-capped PA700-20 S complex. Thus, thedescribed glycerol gradient centrifugation appearsan appropriate method to resolve 26 S proteasomesinto their constituent complexes, yielding a largelyhomogeneous preparation of PA700-20 S complexfor performing reconstitution experiments withpuri®ed PA28 activator and functional studies onthe ensuing hybrid proteasomes.

PA28ab complex, the other reactand, was puri-®ed to apparent homogeneity. When mounted forelectron microscopy (Figure 2(a) and (c)), the acti-vator preparation appears to be devoid of contami-nants. Depending on their orientation, the cup-shaped structures either appear as 10 nm rings inend view, similar to the preparations ®rst reportedby Gray et al.18 or as U-shaped objects in side viewas indicated by arrows in the inset in Figure 2(c).The micrograph in Figure 2(b) is at higher magni®-cation, a further example of peak two proteinfollowing glycerol-gradient centrifugation and cor-responding to the single-capped PA700-20 S com-plex (see also Figure 1(b), peak two).

After mixing of puri®ed PA28 activator andpuri®ed 20 S-PA700 complex, electron micro-scopic analysis of the product (Figure 3(a))revealed an appreciably large number of cork-screw-shaped PA28-20 S-PA700 hybrid com-plexes. Figure 3(b) shows, at 106 foldmagni®cation, an example of hybrid proteasomewith a 20 S core capped at one end by a PA700complex and at the opposite end by a PA28activator complex. The hybrid proteasome has alength of 38 nm as measured along the axis ofthe 20 S core. From the type of micrographshown in Figure 3(a), we collected 220 individ-ual pictures of hybrid complexes and assortedthese into about 20 classes by visual inspection.The pattern obtained collectively showed thathybrid complexes adsorb to the substrate carbon®lm side-on without a preferred orientation ofthe asymmetric PA700 complex, as illustrated bythe gallery of examples from this collection(Figure 3(c)). The particles appear to be caughtas per chance at various stages of rotationaround their long axis while adsorbing to thesubstrate. Barely two identical con®gurations canbe recognized within the collection, precludingconventional image averaging.

In extension of the experiments shown inFigure 1(c), we assessed the relative contribution ofPA700 and PA28ab activator to the activity of theensuing complexes. The results (Table 1) show thatin complexes binding PA28, PA700 or both, 20 Sproteasomal activity is stimulated several fold. Sig-ni®cantly, as seen with the chymotryptic peptidaseactivity (Figure 1(c)), the three complexes displayan almost identical activity toward a given sub-strate, speci®c for the proteasomal chymotryptic,tryptic and postglutamyl peptide hydrolyzingactivity.

Interestingly, as has been reported recently foryeast 20 S proteasome, deletion of the N-terminaltail of the a3 subunit (Y13) which comprises ablocking peptide, opens the central channel with aconcomitant increase in peptidase activity whencompared with that of ``latent'' wild-type 20 S pro-teasomes. The same, higher level of activity ismeasured with PA700-containing 26 Sproteasomes.26,27 Our data for the human PA700-20 S-PA700 (26 S) proteasome complex are inagreement with those authors' ®ndings and extend

Figure 3. Electron micrographs of negatively stained PA28-20 S-PA700 hybrid proteasomes. (a) View of a ®eldshowing the products obtained by reaction of puri®ed PA28 with 20 S-PA700 complex (compare with Figure 2(a) and(b)). Many corkscrew-shaped hybrid proteasomes are detectable. (b) PA28-20 S-PA700 hybrid proteasome at highmagni®cation. The three contributing complexes are indicated. (c) The gallery of nine examples shows hybrid com-plexes which, due to rotation around their long axis, each adsorb to the carbon ®lm support in a different orientation.Compare the different positions of the PA700 moiety at the upper end of the complex by proceeding from left toright in the gallery. Methods: About equimolar amounts of PA28 activator protein and 20 S-PA700 proteasome com-plex (5 mg and 35 mg, respectively) isolated as in Figure 1, were mixed. After overnight standing at 6 �C, the samplewas subjected to electron microscopy as described in the legend to Figure 1.

Hybrid Proteasomes of Human Blood Cells 469

the postulated ``gated channel mechanism'' to

PA28 as a cognate regulatory protein which, by

capping the 20 S proteasome either alone or

together with PA700 in the hybrid proteasome, can

open the central channel to allow free substrate

access.

Table 1. Effect of PA28 on the digestion of ¯uorogenic tripe

20 S (Control) PA28-20Substrate Ac

Suc-LLVY-MCA 130 � 20 1840 �Bz-VGR-MCA 110 � 10 1150 �Z-LLE-MCA 80 � 15 1320 �

A 1.8 nM solution of 20 S, 20 S-PA700 or PA700-20 S-PA700 prothe legend to Figure 1 was preincubated for 15 minutes at room tprotein. The 20 S proteasomes from glycerol gradient peak one (cosubstrate to a ®nal concentration of 100 mM and a further incubareporter group was monitored spectro¯uorimetrically as in the legpendent experiments.

In vivo, the presence of hybrid proteasomescould improve the ef®ciency of a number of pro-cesses mediated by proteasomes. For example,antigen processing which otherwise necessitates atwo-step proteolytic process, initial degradation ofubiquitin-labelled antigen protein by the PA700-20 S proteasome and ®nal trimming by the PA28-

ptides by different proteasome complexes

Proteasome complexS-PA28 PA28-20 S-PA700 PA700-20 S-PA700tivity (pmol/ml per minute)

80 1810 � 100 1820 � 9560 1090 � 80 1120 � 6060 1410 � 90 1450 � 100

tein, obtained after glycerol gradient centrifugation as detailed inemperature in the presence of about equimolar amounts of PA28ntrol) were preincubated with buffer alone. Following addition oftion for 15 minutes at 37 �C, the reaction was stopped and theend to Figure 1. Values given are means � SEM from four inde-

470 Hybrid Proteasomes of Human Blood Cells

20 S proteasome to produce the immunodominantligand could be achieved within the same complex.Furthermore, the complex could couple directly tothe MHC class I peptide loading complex in theendoplasmic reticulum. According to a hypothesisput forward by Li & Rechsteiner,13 this interactioncould be brought about between KEKE motifsexposed on the side distal to the binding surface ofthe PA28a-subunit30 and the KEKE motif contain-ing calnexin which is part of the peptide loadingcomplex in the endoplasmic reticulum (ER)membrane. Thus, free diffusion of the newlytransported peptide and the risk of abortive degra-dation by other cytosolic proteinases would be pre-vented. The reconstitution of hybrid proteasomesdescribed in the present report provides the possi-bility to test this hypothesis.

Acknowledgments

We thank Ms Jutta Kuehn for her skilful technicalassistance. This work was supported by the MinisteriumfuÈ r Wissenschaft und Forschung des Landes Nordrhein-Westfalen, DuÈ sseldorf, and by the BundesministeriumfuÈ r Gesundheit, Berlin.

References

1. Bochtler, M., Ditzel, L., Groll, M., Hartmann, C. &Huber, R. (1999). The proteasome. Annu. Rev.Biophys. Biomol. Struct. 28, 295-317.

2. Kopp, F., Dahlmann, B. & Hendil, K. B. (1993).Evidence indicating that the human proteasome is acomplex dimer. J. Mol. Biol. 229, 14-19.

3. Kopp, F., Kristensen, P., Hendil, K. B., Johnsen, A.,Sobek, A. & Dahlmann, B. (1995). The human pro-teasome subunit HsN3 is located in the inner ringsof the complex dimer. J. Mol. Biol. 248, 264-272.

4. Kopp, F., Hendil, K. B., Dahlmann, B., Kristensen,P., Sobek, A. & Uerkvitz, W. (1997). Subinit arrange-ment in the human 20 S proteasome. Proc. NatlAcad. Sci. USA, 94, 2939-2944.

5. Groll, M., Ditzel, L., LoÈwe, J., Stock, D., Bochtler,M., Bartunik, H. D. & Huber, R. (1997). Structure of20 S proteasome from yeast at 2.4 AÊ resolution.Nature, 386, 463-471.

6. Dahlmann, B., Kopp, F., Kristensen, P. & Hendil,K. B. (1999). Identical subunit topographies ofhuman and yeast 20 S proteasomes. Arch. Biochem.Biophys. 363, 296-300.

7. van Hall, T., Sijts, A., Camps, M., Offringa, R.,Melief, C., Kloetzel, P. M. & Ossendorp, F. (2000).Differential in¯uence on cytotoxic T lympocyte epi-tope presentation by controlled expression of eitherproteasome immunosubunit or PA28. J. Exp. Med.192, 483-494.

8. Larsen, C. N. & Finley, D. (1997). Protein transloca-tion channels in the proteasome and other proteases.Cell, 91, 463-471.

9. Whitby, F. G., Masters, E. I., Kramer, L., Knowlton,J. R., Yao, Y., Wang, C. C. & Hill, C. P. (2000). Struc-tural basis for the activation of 20 S proteasomes by11 S regulator. Nature, 408, 115-120.

10. Coux, O., Tanaka, K. & Goldberg, A. L. (1996).Structure and functions of the 20 S and 26 S protea-somes. Annu. Rev. Biochem. 65, 801-847.

11. Hershko, A. & Ciechanover, A. (1998). The ubiquitinsystem. Annu. Rev. Biochem. 67, 425-479.

12. Yoshimura, T., Kameyama, K., Takagi, T., Ikai, A.,Tokunaga, F., Koide, T. et al. (1993). Molecularcharacterization of the ``26 S'' proteasome complexfrom rat liver. J. Struct. Biol. 111, 200-211.

13. Li, J. & Rechsteiner, M. (2001). Molecular dissectionof the 11S REG (PA28) proteasome activators. Biochi-mie, 83, 373-383.

14. Ahn, K., Erlander, M., Leturcq, D., Peterson, P. A.& FruÈ h, K. (1996). In vivo characterization of theproteasome regulator PA28. J. Biol. Chem. 271,18237-18242.

15. Kuehn, L. & Dahlmann, B. (1996). Reconstitution ofproteasome activator PA28 from isolated subunits:optimal activity is associated with an a,b-heteromul-timer. FEBS Letters, 394, 183-186.

16. Song, X., Mott, J. D., von Kampen, J., Pramanik, B.,Tanaka, K., Slaughter, C. A. & DeMartino, G. N.(1996). A model for the quaternary structure of theproteasome activator PA28. J. Biol. Chem. 271, 26410-26417.

17. Zhang, Z., Krutchinsky, A., Endicott, S., Realini, C.,Rechsteiner, M. & Standing, K. G. (1999). Protea-some activator 11S Reg or PA28: RecombinantRega/Regb heterooligomers are heptamers. Biochem-istry, 38, 5651-5658.

18. Gray, C. W., Slaughter, C. A. & DeMartino, G. N.(1994). PA28 activator protein forms regulatorycaps on proteasome stacked rings. J. Mol. Biol. 236,7-15.

19. Groettrup, M., Soza, A., Eggers, M., Kuehn, L., Dick,T. P., Schild, H. et al. (1996). A role for the protea-some regulator PA28alpha in antigen presentation.Nature, 381, 166-168.

20. Dick, T. P., Ruppert, T., Groettrup, M., Kloetzel, M.,Kuehn, L., Koszinowski, U. H. et al. (1996). Coordi-nated dual cleavages induced by the proteasomeregulator PA28 lead to dominant MHC ligands. Cell,86, 253-262.

21. Preckel, T., Fung-Leung, W. P., Cai, Z., Vitiello, A.,Salter-Cid, L., Winqvist, O. et al. (1999). Impairedimmunoproteasome assembly and immuneresponses in PA28ÿ/ÿ mice. Science, 286, 2162-2165.

22. Kuehn, L. & Dahlmann, B. (1996). Proteasomeactivator PA28 and its interaction with 20 Sproteasomes. Arch. Biochem. Biophys. 329, 87-96.

23. Adams, G. M., Crotchett, B., Slaughter, C. A.,DeMartino, G. N. & Gogol, E. P. (1998). Formationof proteasome-PA700 complexes directly correlateswith activation of peptidase activity. Biochemistry,37, 12927-12932.

24. Hendil, K. B., Khan, S. & Tanaka, K. (1998). Simul-taneous binding of PA28 and PA700 activators to20 S proteasomes. Biochem. J. 332, 749-754.

25. Tanahashi, N., Murakami, Y., Minami, Y., Shimbara,N., Hendil, K. B. & Tanaka, K. (2000). Hybrid pro-teasomes. Induction by interferon-g and contributionto ATP-dependent proteolysis. J. Biol. Chem. 275,14336-14345.

26. Groll, M., Bajorek, M., KoÈhler, A., Moroder, L.,Rubin, D. M., Huber, R., Glickman, M. H. & Finley,D. (2000). A gated channel into the proteasome coreparticle. Nature Struct. Biol. 7, 1062-1067.

27. KoÈhler, A., Bajorek, M., Groll, M., Moroder, L.,Rubin, D. M., Huber, R., Glickman, M. H. & Finley,

Hybrid Proteasomes of Human Blood Cells 471

D. (2001). The substrate translocation channel of theproteasome. Biochimie, 83, 325-332.

28. Hough, R., Pratt, G. & Rechsteiner, M. (1987). Puri®-cation of two high molecular weight proteinasesfrom rabbit reticulocyte lysate. J. Biol. Chem. 262,8303-8313.

29. Dahlmann, B., Kuehn, L. & Reinauer, H. (1995).Studies on the activation by ATP of the 26 S

proteasome complex from rat skeletal muscle.

Biochem. J. 309, 195-202.

30. Knowlton, J. R., Johnston, S. C., Whitby, F. G.,

Realini, C., Zhang, Z., Rechsteiner, M. & Hill, C. P.

(1997). Structure of the proteasome activator Rega

(PA28a). Nature, 390, 639-643.

Edited by R. Huber

(Received 6 June 2001; received in revised form 31 August 2001; accepted 3 September 2001)