Identification and Characterization of Three Different Subpopulations

Vol. 264, No.12, Issue of April 25, pp. 6660-6666.1989

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Societyfor Biochemistry and Molecular Biology, Inc.

Printed in U.S.A.

Identification and Characterization of Three Different Subpopulations of the Drosophila Multicatalytic Proteinase (Proteasome)” (Received for publication, October 26,1988)

Patricia-E. FalkenburgS and Peter“. Kloetzelg From ZMBH/Molekulare Genetik, Im Neuenheimer Feld 282,6900 Heidelberg, Federal Republic of Germany

In Drosophila melanogaster the population of pro- indicate a highly important physiological function of the teasome particles consists of three distinct subclasses. particle. Despite this, the in uiuo substrates and the regulation By fractionation of a 40,000 X g supernatant of Dro- of the activity of the proteasome are still unknown. sophila homogenate on a DEAE-Sephacelcolumn, In Drosophila the proteasome appears t o be localized preproteasome particles which elute at salt concentra- dominantly in the cytoplasm (14,15) and is in part associated tions of 200, 300, and 500 mM KAc can be separated. with polysomal fractions (16). The variability of the proteaThe proteasomes of all three subfractions sediment at some protein composition during the development of the fly 19 S in sucrose gradients and are shown by two- (17) and the differential cellular distribution of the particle dimensional gel electrophoretic analysis to possess in several species led us to investigatewhether the proteasome the same protein content, They differ, however, with respect to their specific proteolytic activity against population can be separated into different subclasses that thesubstrates benzoyl-Val-Gly-Arg-4-methylcou-might differ in biochemical composition, enzymatic activity, maryl-7-amide, succinyl-Leu-Leu-Val-Tyr-4-methyl-and biological function. coumaryl-7-amide, and succinyl-Ala-Ala-Phe-4-methMATERIALS ANDMETHODS ylcoumaryl-7-amide and the degree to which their hydrolytic activity can be enhanced by the addition of Preparation of Cytoplasmic Extracts-For the preparation of cyto30-110 I.~Msodium dodecyl sulfate (SDS). Our data plasmic extracts adult flies were homogenized in 80 mM KAc buffer show that the 200 mM proteasome fraction exhibits the (80 mM KAc, 15 mm MgAc2,lOmM Hepes,’ pH 7.2). The homogenate lowest basal specific proteolytic activity but can be was centrifuged for 10 min to prepare a 40,000 X g supernatant stimulated most by SDS. The 300 and 500 mM protea- (SOL80 extract). This homogenate was either passed through 0.45some subfractions, on the other hand possess consid- pm membrane filters (Schleicher & Schuell) and applied to a DEAESephacel-column or loaded on 10-40% sucrose gradients (centrifuerably higher but similar basal specific proteolytic ac- gation to 02t= 1,10’*radiansz/s, 4 “C toisolate proteasome particles). tivity. Of these only the proteolytic activity of the 300 Isolation of a Total Population of Proteasome Particles-A total mM subfraction against the substrates benzoyl-Val- population of proteasome particles can be isolated from SOL80 exGly-Arg-4-methylcoumaryl-7-amide and succinyl- tracts by two subsequent steps of sucrose gradient centrifugation and Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide can be extraction with ‘‘high salt buffer” (0.5 M KAc, 15 mM MgAcz, 10 mM Hepes, pH 7.2) as described previously (14). enhanced by SDS. DEAE-Sephacel Column Chromatography-DEAE-Sephacel Our data raise the possibility that the different subpopulations reflect structural differences between the (Pharmacia LKBBiotechnology Inc.) was equilibrated in 80 mM KAc proteasome particles, which in turn may result in dif- buffer. SOL80 extract was then incubated with the matrix material for 2 h. The matrix was washed batchwise with 80 mM KAc buffer ferent in vivo substrate specificities of the proteasome until all unbound material was removed, as judged by measurement subpopulations. of the AzWof the supernatant. After this, a column was formed and the bound material eluted with 200, 300, and 500 mM KAc buffer (KAc in the indicated concentration, 15 mM MgAcz, 10 mM Hepes, pH 7.2) in steps. Elution with the next higher salt buffer was only In two previous reports (1,2) i t has been shown that the started when the AZw in the eluting fractions wasbelow0.3. The material was collected in 1-or 5-ml fractions. “19 S cylinder-type particle” or “prosome” and a high molecAssay of Proteolytic Actiuity-To test whether protein samples ular weight proteinase complex are identical structures. This were proteolytically active three different fluorogenic substrates were proteolyticallyactive,multicatalyticcomplex,“the protea- used (1 or 10 HM of Bz-Val-Gly-Arg-MCA,Suc-Leu-Leu-Val-Tyrsome,” has been isolated from organismsas widely separated MCA, and Suc-Ala-Ala-Phe-MCA; seeRef. 1). The samples were in evolution as plants, yeast, Drosophila, Xenopus, and hu- incubated in 1 ml of reaction buffer (10) at 37 “C. The reaction was mans (see, for example, Refs. 4-13 and, for a review, Ref. 3). either monitored continuously to study the effect of SDS addition or after 30 minof incubation by addition of 100 pl of stop buffer In all cases it is characterized by a typical cylinder-shaped stopped (18). The reaction was measured at an emission wavelength of 480 morphology and a protein composition of 12-25 polypeptides nm using a spectrofluorometer RF-5000, Shimadzu, with excitation i n t h esize range of 19-35 kDa. The ubiquityand evolutionary a t 366 nm. The specific proteolytic activity of each sample was conservation of this complex are t h u s well documented and calculated as the increase in fluorescence at 480 nm/pg of protein/ min. One- and Two-dimensional Gel Electrophoresis-Protein samples * This work wassupported in part by Deutsche Forschungsgemeinschaft Grant SFB 229 Kl/C4. Thecosts of publication of this article were electrophoretically separated according to theprotocols of Laemwere defrayed in part by the payment of page charges. This article mli (19) and Studier (20) for one-dimensional SDS-polyacrylamide must therefore be hereby marked “aduertisement” in accordance with gel electrophoresis and the protocol of OFarrell (21) for two-dimensional isoelectric focusing. 18 U.S.C. Section 1734 solelyto indicate this fact. $Supported by a Ph.D. grant from the Fonds der Chemischen The abbreviations used are: Hepes, 4-(2-hydroxyethyl)-l-piperaIndustrie. § T o whom correspondence should be addressed. Tel: 06221- zineethanesulfonic acid; Bz, benzoyl; SUC,succinyl; MCA, &methylcoumaryl-7-amide; SDS, sodium dodecyl sulfate. 566853.

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6660

Subpopulations of the Drosophila Proteasome

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FIG. 1. A cytoplasmic supernatant (SOLSO) of adult Drosophila melanogaster was fractionated on a DEAE-Sephacel column. Todetermine whether all proteasome particles containedin the SOL80 extract bind to the column under the conditions used, the extract was loaded on the preformed column and fractions were collected during the wash with 80 mM KAc buffer. Thus, in contrast to theprocedure described under "Materials and Methods," a batch procedure was not used in the experiment shown. Bound material was eluted with 200,300, and 500 mM KAc buffers (see "Materials and Methods"). a, absorption profile of the DEAE-Sephacel column, absorbance ( O D ) monitored a t 280 nm. The protein content of the column fractions was determined by SDSpolyacrylamide gel electrophoresis (12% gel), and the proteins were subjected to immunoblotting using a mixture of the antibodies Dm35K1, Dm28K2, Dm25K1, and Dm23K1. These antibodies react specifically with the protein subunits of the Drosophila proteasome (13). Proteasome-specific proteins are present in all three salt fractions. b, the proteolytic activity of the 200,300, and 500 mM fractions was determined using the fluorogenic substrates BzVal-Gly-Arg-MCA (-), Suc-Leu-Leu-Val-Tyr-MCA (- - - -), and Suc-Ala-Ala-Phe-MCA (.-.-); the substrate concentration was always 1PM.

500 mM KAc (Fig. la) and the proteolytic activity of these fractionsagainst Bz-Val-Gly-Arg-MCA, Suc-Leu-Leu-ValTyr-MCA, and Suc-Ala-Ala-Phe-MCA was determined (Fig. Ib). step-elution with IUc-buffers The protein contentof the fractions was analyzed on 12% SDS-polyacrylamide gel electrophoresis, and proteasome pro200 mU-FRACTION 300 lan-FRACTION 500 mU-FRACTION teins were identified by immunoblottingusinga mixture of the proteasome-specific antisera Dm35K1, Dm28K2, Dm25K1, and Dm23K1 (14). F I R S T 10-40 % SUCROSEGRADIENT, As shown in Fig. la, proteasomal proteins can be demonstrated in fractions eluted with 200, 300, and 500 mM KAc 19s-fractions pslleted and reextraction of the pellets in 0.5 I4 IUC-buffer buffer. Elution with salt gradients or with narrower steps of KAc buffer gave similar results (data notshown). SECOND 10-40 % SUCROSE GRADIENT, 0 . 5 n KAc-BUFFER The proteolytic activity of the column fractions was tested 1 using the fluorogenic substrates Bz-Val-Gly-Arg-MCA, SUC200 mU-SUBPOPULATION 300 mU-SUBPOPUIATION 500 mU-SUBPOPLIUTION Leu-Leu-Val-Tyr-MCA, and Suc-Ala-Ala-Phe-MCA (Fig. Suc-Ala-Ala-Phe-MCA ishydrolyzed most FIG. 2. Purification scheme forthe isolation of proteasome lb). The substrate (see effectively by the 200 mM fraction, while the proteolytic particles from all three DEAE-Sephacel column fractions text). activity which hydrolyzes Suc-Leu-Leu-Val-Tyr-MCA peaks in the 200 and 500 mM fractions withsomeactivityalso Immunoblotting-Analysis of electrophoreticallyseparatedpropresent in the 300 mM salt eluate. The proteolytic activity teins by immunoblotting was performed as described previously (14). against Bz-Val-Gly-Arg-MCA in all fractions tested is much higher than that against the other two substrates. Itis hydroRESULTS lyzed best by the material eluting in 500 mM KAc from the Previously it was shown that theproteasomes of Drosophila DEAE-Sephacel column. For comparison of the biochemical and enzymatic characcan be isolated from the soluble cytoplasmic 40,000 X g different salt fractions supernatant of cellular extracts, i.e. SOL80 extract (16). To teristics of the proteasomes in the three test whether the apparently homogeneous proteasome popu- they had to be purified further. To be able to exclude the lation can be further fractionated, SOL80 extract of adult possibility that differing properties of the subpopulations are flies was subjected to DEAE-Sephacel column chromatogra- due to differences in their preparation, we took great care the phy. Bound material was eluted by salt stepsof 200,300, and that all three proteasomalsubpopulationsunderwent CYTOPLASWIC SUPERNATANT

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FIG. 3. Proteasome-containing fractions of the 200, 300. and 500 mM DEAE-Sephacel column eluates were pooled separately and, after dialysis and concentration, subjected to centrifugation on 10-40% sucrose gradients in 80 mM KAc buffer. I, 200 mM subpopulation. a, absorption profile of the sucrose gradients. b, the protein content of the sucrose gradient fractions was analyzed on 12%SDS-polyacrylamide gels. For electrophoresis, two adjacent fractions of the gradients were always pooled. Proteasome-specific proteins sediment in gradient fractions 11-12 and aremarked with arrows; lane M , marker. c, the proteolytic activity of the Suc-Leu-Leu-Val-Tyr-MCA (- - - -), and Sucgradient fractions was determined Bz-Val-Gly-Arg-MCA (-); . 300 mM subpopulation. a, b, and c are Ala-Ala-Phe-MCA (-.-.); the substrate concentrationwas always 1 p ~II, as described for I. III, 500 mM subpopulation. a, b, and c are as described for I.


6663

same treatment during purification (Fig. 2). Therefore, the lyzing activity was found sedimenting between 15 and 18 S DEAE-Sephacel column fractionswere pooled separately and which is not associated with theproteasome. concentrated, as well as adjusted to 80 mM KAc by vacuum Likewise, the distributionof the Bz-Val-Gly-Arg-MCA and dialysis. The 200, 300, and 500 mM fractions were then Suc-Ala-Ala-Phe-MCA degrading activities in the gradient 80 mM KAc buffer fractionsvaries considerably and is notrestrictedtothe centrifuged on10-40% sucrose gradients in (first gradient). The 19 S fractions of these gradients were proteasome fractions of the gradients (Fig. 3, IC,IZc, ZZIc). In pelleted, the pellets extracted with 500 mM KAc buffer, and particular, hydrolysis of the Bz-Val-Gly-Arg-MCA substrate recentrifuged on a second sucrose gradient in 500 mM KAc is not maximal in the proteasome fractions of the gradients. buffer. Thus, all threeproteasomalsubpopulations were These data show that the proteasomes, which are enriched in treated with the same buffers. the 19 S fraction of the sucrose gradients, are still contamiThe protein composition and proteolytic activity of the nated by other cellular components. These contaminations, gradient fractions of both sucrose gradient steps were ana- however, can be removed almost completely by extraction of lyzed. the 19S material with 0.5 M KAc buffer and recentrifugation As shown in Fig. 3, la, IZa, ZZZa, and Zb, ZIb, ZZIbproteasomes on a second sucrose gradient (Fig. 4). Analysis of the protein which sediment at 19 S can be isolated from all three DEAE- composition of the 19S peak fractionsof these second sucrose Sephacel column fractions. Immunoblot experiments using gradients demonstrates that almostall contaminating matethe proteasome-specific antiserum Dm 19sc (1)showed that rial has been removed from the proteasomes present in the no particles are present in the pellets of these gradients (data 300 and 500 mM KAc column fractions (Fig. 4d). not shown). The specific proteolytic activity against Suc-Leu- TheproteolyticactivityagainstSuc-Leu-Leu-Val-TyrLeu-Val-Tyr-MCA is strongly enriched in the 19 S fraction MCA and Suc-Ala-Ala-Phe-MCA is strongly enriched in the IIc, IIZc). There also proteasome fractionsof these second sucrose gradients.Howof the 500 mM subpopulation (Fig. 3, IC, is a peak of this activity in the 19 s sucrose gradient fractions ever, Suc-Ala-Ala-Phe-MCA is cleaved less efficiently by the of the 200 and 300 mM subpopulations (Fig. 3, IC, ZIc, IIIc). proteasomethan Suc-Leu-Leu-Val-Tyr-MCA. Inaddition, This activity, however, is less marked, and in the 200 mM the Bz-Val-Gly-Arg-MCA cleaving activity appears tobe fursubfraction a very strong Suc-Leu-Leu-Val-Tyr-MCA hydro- ther enriched in the proteasome fractions, but less dramatically (Table I). Using these substrates no proteolytically other active complexes can be detected in these gradients. a b Comparison of the specific proteolytic activity of all proteasome containing fractions during the purification procedure (TableI) shows thatenrichmentand purification of the proteasomes are accompanied by a strong increasein the specific proteolytic activity. It is evident, however, that the three proteasome subpopulations differ in their proteolytic efficiency. In particular, the 200 mM proteasome subpopulation appears tobe rather inactive under the conditions used. The difference in the affinityof the proteasomal subpopulations to the anion-exchange matrix may be due to a difference in the charge of the complexes. Such a difference may be caused by a slightly altered subunit composition of the C enzyme or by the modification of certain proteincomponents. The molecular composition of the different proteasome subpopulations was therefore carefully compared. The proteasomal proteins were separated by two-dimensional gel electrophoresis, and the resulting patterns were analyzed (Fig. 5 ) . No major qualitative differences in the protein pattern aredetectable. Slight variations in the protein pattern observed between different experiments (forexample spot 1, spot 17, and the spot marked with arrow) an were not in all cases reproducibleand thus are not regarded as specific. v, I i'4'6'B'lb'i2'1'4'1~'1~'2b Differences in the relative amountof certain protein compofractions d nents cannot be entirely excluded, but due to the methods m m M 300rnM 5oomM used cannot be unequivocally proven. It is evident, however, 9 10 11 12 1319 10 11 12 1319 x) 11 12 13 that the difference in the elution behavior of proteasomal subpopulationsfromthe DEAE-Sephacelcolumn isnot caused by a drastic difference in their subunitcomposition or varying modificationsof the protein component of the proteasomes. As shown above, the proteasomal subpopulations, as defined by their elution behavior from DEAE-Sephacel, differ a t least in part in theirspecific proteolytic activity (Table I). FIG. 4. The 19 S fractions of the firstsucrose gradients (see One of the main characteristics of the proteasomalproteolytic Fig. 2) were pelleted and after extraction with 0.5 M KAc activity is its enhancability by small amounts of SDS (see, buffer recentrifuged on second 10-40% sucrose gradients. for example, Ref. 22). Therefore, the effect of SDS on the The absorption profiles of these second gradients are shown. a, 200 proteolytic activity of the 200, 300, and 500 mM KAc proteamM subpopulation, b, 300 mM subpopulation and c, 500 mM subpopulation. d , the protein content of the 19 S peak fractions of these some subpopulations was analyzed. As shown in Fig. 6 and gradients was analyzed on a 12% SDS-polyacrylamide gel. Table 11, there exist marked differences between the protea-


6664

TABLE I Proteasome subfractionswere isolated and purified as described above (seeFig. 2) The specific proteolytic activity of all proteasome containing fractions arising during this purification procedure , (1 pM), was compared. Substrates used were Bz-Val-Gly-Arg-MCA (AMC) (1 p ~ ) Suc-Leu-Leu-Val-Tyr-MCA and Suc-Ala-Ala-Phe-MCA (1 UM).

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activity of the 200 mM subpopulation against the substrate Suc-Ala-Ala-Phe-MCA reaches the same level as that of the 300 mM subpopulation against the same substrate without SDS. While the proteolytic activity of the 300 mM subpopulation against Suc-Ala-Ala-Phe-MCA is not enhanced by SDS, its hydrolytic activity against Suc-Leu-Leu-Val-Tyr-MCA and Bz-Val-Gly-Arg-MCA is enhanced 2-3.4-fold. Interestingly, this subpopulation reaches its maximal activity against the substrate Bz-Val-Gly-Arg-MCA at an SDS concentration of 90 p ~ a concentration , that is markedly higher than that required for the maximal activation of the 200 mM subpopulation againstthe same substrate.The 500 mM subpopulation, on the other hand, which possesses a high basal specific proteolytic activity against all three substrates cannot be activated. The proteolytic activity of all three proteasomal subpopulations is inhibited when the SDSconcentration is raised over a critical value, whereby the amount of SDS required for inhibition is different for each substrate andfor each subpopulation (Fig. 6).

-36 DISCUSSION

-29

Our data show that by using DEAE-Sephacel column chromatography as an initial purification step, three proteasomal subpopulations can be distinguished which elute at saltconcentrations of 200, 300, and 500 mM KAc. This differential FIG. 5. Proteasome particles of the 200, 300,and 500 mM DEAE-Sephacel column eluates were isolated and purified as elution appears to be quite unusual in that all proteasome described above. The protein composition of these subpopulations purification procedures published so far which involve the was compared after two-dimensional gel electrophoretic separation. DEAE-column step show that thisproteolytically active, mulThe spots were numbered accordingto Haass and Kloetzel (17). tifunctional complex elutes as a single peak a t salt concentrations between 230 and 300 mM depending on the tissue and the purification method used (for example, Refs. 7, 13, 23, some fractions with regard to their sensitivity toward SDS and 24). treatment. The proteasomes of all three subfractions possess identical The specific proteolytic activity of the 200 mM subpopulas values and a similar proteincomposition. Despite this, they tion against the peptide substrates used is strongly increased in the presence of SDS. However, the amount of SDS which are notonly distinguishable by their chromatographic behavis necessary for maximal activation of the proteolytic activity ior but also with regard to theirenzymatic activity. The three is different for the threesubstrates, i.e. 50 p~ for Bz-Val;Gly- proteasomal subpopulations differ in their specific proteolytic Arg-MCA,30 p~ for Suc-Leu-Leu-Val-Tyr-MCA, and 110 activity against the fluorogenic peptide substrates Suc-AlaAla-Phe-MCA, Suc-Leu-Leu-Val-Tyr-MCA,and Bz-Val-Glyp~ for Suc-Ala-Ala-Phe-MCA. While the basal specific proteolytic activity of the 200 mM Arg-MCA, as well as with respect to their susceptibility to subpopulations is very low and relatively large amounts of SDS treatment. The 200 mM subpopulation possesses by far the lowest enzyme are needed to monitor an enzyme reaction, SDS stimulates the proteolytic activity against the substrateSuc- basal specific proteolytic activity of the three proteasome Leu-Leu-Val-Tyr-MCA to a level which is similar to that of subpopulations. On the otherhand, this is also the proteasome the 500 mM subpopulation against the same substrate without fraction whose proteolytic activity is enhanced most by SDS. SDStreatment. Similarly, the SDS-stimulated proteolytic The 300 and 500 mM subpopulations both possess signifi-

Subpopulations of the Drosophila Proteasome k-V-G-R-MCA

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activities of the200 (-), 300 (- - -), and 500 mM (-. - .) proteasome subpopulations against the substrates Bz-Val-Gly-Arg-MCA (1 PM), Suc-Leu-Leu-Val-Tyr-MCA (1 p ~ )and , Suc-Ala-Ala-Phe-MCA (10 p ~before ) and after addition of SDS were compared. SDS was added to the incubation mixture in the indicated amounts andthe reaction was measured continuously (see "Materials and Methods"). The dependence of specific proteolytic activity of the enzyme particles on SDSconcentration was calculated as Am unitslpg protein and per min.

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cantly higher basal specific proteolytic activities. Nevertheless, they differ in thatonly the proteolytic activity of the 300 mM subpopulation can be enhanced by SDS. Thus, the 200 mM subpopulation might represent an inactivated or latent form of the enzyme which by some up to now unknown mechanism can be transferred into the activated state, i.e. the 300 and 500 mM subpopulations. On the other hand, the subpopulations may bepresent inthe cell in amore or less latent state and require the binding of a specific substrate for the activation of their proteolytic activity. For the interpretation of the data presented here it is important to note that all three proteasome fractions underwent the same purification and extraction procedure after the initial DEAE-Sephacel column step. Thus, all three subfractions were exposed to the same high salt treatment, and it therefore appears unlikely that the differences in their enzymatic activities are the result of an experimentally induced removal of loosely bound factors which either activate or inactivate the enzyme. An open question at present is why the differences in the enzymatic properties of the proteasome subpopulations coincide with a difference in chromatographic behavior. Coelution with other cellular components appears very unlikely since a total population of purified 19 S proteasome particles still separates into three subpopulations.* One possible explanation is that the presence of different subpopulations reflects a difference in the three-dimensional structure of the proteasome. This would imply that charged groups of the proteasomal protein subunits may become exposed on the outer surface of the complex and thus be more available for interaction with the ion-exchange matrix. Along

* P.-E. Falkenburg and P.-M. Kloetzel, unpublished observation.

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this line of argumentation, the active site for the substrate Suc-Leu-Leu-Val-Tyr-MCA in the 500 mM subpopulation wouldbe present in an open conformation, allowingeasy binding of the substrate, while in the 300 mM subpopulation it would be partially obscured, and in the 200 mM subpopulation nearly unavailable. Thus, the enzymatic differences between the threesubpopulations could reflect specific differences in the in vivo substrate specificity of the threetypes of proteasome particles. A number of data suggest a tissue- and developmentally specific regulation of proteasome activity. For sea urchin proteasome particles it was shown that they are distributed in a developmentally dependentmanner between "free" fractionsand fractions of higher molecular weight that are associated with nuclear material (25). Kuehn et al. (26) demonstrated different specific activities for MCP particles isolated from rat reticulocytes and erythrocytes. In Drosophila, the two-dimensional protein pattern of the proteasome is altered in developmentally a dependent manner (17). In summary, the proteasome functions may be of essential importance for post-translational modification, processing of developmentally or tissue-specific proteins, or the regulation of their turnover. Of course, these conclusions are still speculative and careful analysis of the in vivo substrate specificity of the proteasome will be necessary to elucidate the biochemical role of this complex. Acknowledgments-We wish to thank Professor E.K. F. Bautz for providing excellent working conditions and Ch. Haass and A. Seelig for many helpful discussions.

1.

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14. Schuldt, C., and Kloetzel, P.-M. (1985) Deo. Biol. 110,65-74 15. Arrigo, A.-P., Darlix, J.-L., Khandjian, E.W., Simon, M., and Spahr, P.-F. (1985) EMBO J. 4, 399-406 16. Kloetzel, P.-M., Falkenburg, P.-E., Hossl, P., and Glatzer, K.H. (1987) Exp. Cell Res. 170, 204-213 17. Haass, C., and Kloetzel, P.-M. (1989) Exp. CellRes. 180, 243252 18. Barrett, A. J. (1980) Biochem. J. 187,909-912 19. Laemmli, U. K. (1970) Nature 227,680-685 20. Studier, W. F. (1975) J. Mol. Biol. 98, 503-517 21. O’Farrell, P. H. (1975) J. Bwl. Chem. 250,4007-4021 22. Dahlmann, B., Rutschmann, M., Kuehn, L., and Reinauer, H. (1985) Biochem. J. 228, 171-177 23. Dahlmann, B., Kuehn, L., Rutschmann, M., and Reinauer, H. (1985) Biochem. J. 228,161-170 24. Kleinschmidt, J. A,, Hugle, B., Grund, C., and Franke, W.W. (1983) Eur. J. CellBiol. 32, 143-156 A. A. (1987) Proc. 25. Akhayat, O.,Grossi de Sa, F., and Infante, Natl. Acad. Sci. U. S. A. 84, 1595-1599 26. Kuehn, L., Dahlmann, B., and Reinauer, H. (1988)in Intracellular Protein Catabolism. (Katenuma, N., ed) Springer-Verlag, Toin press