THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 31. Issue of November 5, PP. 1620!3-16217,1988 Printed in U.S.A.

Proteasomes (Multi-protease Complexes) as 20 S Ring-shaped Particles in a Variety of Eukaryotic Cells*

(Received for publication, May 9, 1988)

Keiji TanakaS, Tetsuro Yoshimura, Atsushi Kumatori, and Akira Ichihara From the Institute for Enzyme Research, The University of Tokushima, Tokushima 770, Japan

Atsushi Ikai and Masaaki Nishigai From the Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

Keiichi Kameyama and Toshio Takagi From the Institute for Protein Research, Osaka University, Suita 565, Japan

Latent multicatalytic protease complexes, named proteasomes, were purified to apparent homogeneity from various eukaryotic sources, such as human, rat, and chicken liver, Xenopus laevis ovary, and yeast (Saccharomyces cerevisiae), and their functional and structural properties were compared. They showed la- tency in breakdown of [methyLSH]casein, but were greatly activated in various ways, such as by addition of polylysine. They all degraded three types of fluoro- genic oligopeptides at the carboxyl side of basic, neu- tral, and acidic amino acids, and the three cleavage reactions showed different spectra for inhibition, sug- gesting that they had three distinct active sites. The proteasomes all seemed to be seryl endopeptidases with similar pH optima in the weakly alkaline region. Their physicochemical properties, such as their sedimenta- tion coefficients (19 S to 22 S ) , diffusion coefficients (2.0-2.6 X lo-’ cm2 s-’), molecular masses (700-900 kDa), and circular dichroic spectra, were similar. Their amino acid compositions were also very similar. Electron microscopy showed that they had similar well-defined symmetrical morphology, appearing to be ring-shaped particles with a small hole in the center. All the proteasomes seemed to be multisubunit com- plexes consisting of 15-20 polypeptides with molecu- lar masses of 22-33 kDa and isoelectric points of pH 3-10, but they showed species-specific differences in subunit multiplicity. Moreover, they differed immu- nologically, as shown by Ouchterlony tests and immu- noblotting analyses, although cross-immunoreactivi- ties of some subunits or domains were observed. These results indicate that the sizes and shapes of these pro- teasomes have been highly conserved during evolution, but that they show species-specific differences in im- munoreactivities and subunit structures. Thus protea- mmes with similar structure and function seem to be ubiquitously distributed in eukaryotic organisms rang- ing from man to yeast. This distribution implies the general importance of these proteasomes for proteoly- sis.

* This work was supported in part by grants from the Ministry of Education, Science, and Culture of Japan and the Foundation for Enzyme Application, Osaka. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘‘advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed.

Recently we (1, 2) and others (3-17) have reported very large intracellular proteases in various mammalian cells. Studies have shown that these large proteases have quite different functional and structural characteristics from other well-known proteases. These large proteases have some com- mon properties, such as a latent form and multiple peptidase activities that are catalyzed by at least three independent active sites within a single complex (1, 8, 11). Furthermore, these enzymes are all unusually large, with a sedimentation coefficient of approximately 20 S and a molecular mass of 700-800 kDa (2, 18) and consist of multiple nonidentical subunits (19). By electron microscopy they appear to have a symmetrical ring shape (2, 18, 19) that is consistent with a simulated model obtained by x-ray small-angle scattering analysis (19). Based on these findings, we have named these large multifunctional protease complexes proteasomes (19, 20).

Previously (I), we found by quantitative enzyme immu- noassay that these proteasomes were abundant in all rat cells and tissues examined. Although these multicatalytic proteases have been found mainly in various mammalian cells (1,8, ll), similar types of large alkaline proteases have also been found in fish muscle (21, 22) and yeast (23). Therefore, one way of studying the biological role of these proteases is to determine their distribution. In this work, we purified similar types of multi-protease complexes from human, rat, and chicken liver, Xenopus laevis ovary, and yeast, and demonstrated that they are functionally and structurally related enzymes. These find- ings indicate that proteasomes are ubiquitously distributed as large ring-shaped 20 S particles in a variety of eukaryotic cells ranging from human to yeast cells.

Interestingly, the size, shape, and subunit multiplicity of these proteasomes are remarkably similar to those of the ring- shaped particles of 20 S found previously in a wide variety of eukaryotic organisms (24-38), including ribonucleoprotein particles with specific RNA species (24-27, 30-33, 38), 19 S particles with certain heat-shock proteins (25, 26), and par- ticles with tRNA processing nuclease activities (29). Very recently we found that 19 S particles in HeLa cells are identical to mammalian proteasomes (20). Moreover, the identity of 19 S ribonucleoprotein particles with a multicatal- ytic proteinase from rat muscle was reported by Falkenburg et al. (39). However, it is unknown whether these particles are all identical, since these miniparticles differ in immuno- logical reactivities and subunit compositions (31, 38). In this paper, we show that the ubiquity and interspecies structural variations of proteasomes account for these immunological

16209

16210 The Proteasomes (Eukaryotic Multi-protease Complexes)

and structural differences of the various 20 S particles re- ported so far, and discuss the identity of proteasomes with the subcellular 20 S particles in various eukaryotic cells.

EXPERIMENTAL PROCEDURES

Materials-The compounds used were as follows: Fast Flow Q- Sepharose, Mono-Q packed columns (Pharmacia LKB Biotechnology Inc.), Cosmosil5C4-300 packed column (Nakara Chemicals, Kyoto), Suc'-Leu-Leu-Val-Tyr-MCA (Peptide Institute, Inc., Mino), Cbz- Ala-Arg-Arg-MNA and Cbz-Leu-Leu-Glu-NA (gifts from Dr. L. Wax- man), "'I-protein A (8.3 pCi/rg, Du Pont/New England Nuclear), molecular weight markers (Pharmacia), and PI markers (Oriental Yeast Co., Tokyo).

Assay of Protease Activity-Protease activity was assayed as de- scribed previously (1). The degradation of [meth~l-~HIcasein was determined by measuring the radioactivity of the acid-soluble frag- ments cleaved. The hydrolytic activities on various fluorogenic sub- strates were determined by measuring the fluorescence of groups liberated from these peptides.

Purification of Proteasomes from Various Eukaryotic Cells-Pro- teasomes from various sources, such as human, rat, and chicken liver, X. &vis ovary, and yeast were purified by sequential chromatogra- phies on Fast Flow Q-Sepharose, Bio-Gel A-1.5m, hydroxylapatite, heparin-Sepharose CL-GB, and FPLC Mono-Q columns. The proce- dures used were essentially similar to those reported previously (I), but two steps were improved: Fast Flow Q-Sepharose chromatography and FPLC with a Mono-Q column were used as the first and final steps, respectively. Details of the modifications of the procedure are as follows. Fast Flow Q-Sepharose chromatography was carried out a t a high flow rate (200 ml/h). The crude extract was applied to a column (5 X 60 cm) equilibrated with 25 mM Tris-HC1 buffer (pH 7.5) containing 1 mM 2-mercaptoethanol and 20% glycerol, and proteins were eluted with a linear gradient of 0-0.8 M NaCI. Fractions with high activity were concentrated by ultrafiltration on a PM-10 membrane (Amicon) to about 25 ml and then applied to a Bio-Gel A- 1.5m molecular sieving column. The enzymes from X. lueuis and yeast were concentrated as described above, whereas those from other sources were concentrated by precipitation with polyethylene glycol 6000 (15%), because the protein concentration was higher. Subse- quent steps on Bio-Gel A-1.5m, hydroxylapatite, and heparin-Seph- arose CL-GB columns were performed as described previously (1). A Pharmacia FPLC system with a Mono-Q column was used as the final step of purification of all these enzymes. The enzyme prepara- tions obtained by heparin-Sepharose CL-GB chromatography were directly subjected to Pharmacia FPLC on a Mono-Q column (1 X 10 cm) equilibrated with 25 mM Tris-HC1 buffer (pH 7.5) containing 1 mM dithiothreitol and 20% glycerol. Elution was carried out at a flow rate of 1 ml/min with a linear gradient of 0-0.8 M KC1 in the same buffer. The enzymes from human, rat, chicken, and Xenopus were eluted from the column as single symmetrical peaks at the same concentration of about 340 mM KCl, whereas the enzyme from yeast was eluted at a higher KC1 concentration (400 mM). This procedure was completed within 2 h at room temperature, and all other proce- dures were performed at 4 "C.

Starting with 100 g (wet weight) of tissues from various sources, averages of 5.1 mg (human liver), 4.5 mg (rat liver), 2.6 mg (chicken liver), 2.9 mg (X. luevis ovary), and 3.1 mg (yeast) of purified enzymes were obtained. The final enzyme preparations were apparently ho- mogeneous, judging by their single symmetrical profiles on Mono-Q FPLC and their single protein bands on electrophoresis in polyacryl- amide gel under nondenaturing conditions.

Biochemical Analyses-SDS-PAGE was carried out by the method of Laemmli (40) in 12.5% slab gels with or without 0.1% SDS. Isoelectric focusing in polyacrylamide gel with urea was done by the method of O'Farrell (41), and that in a column according to the manufacturer's protocol. Proteins were detected with Coomassie Bril-

phosphorylase b (94,000), BSA (67,000), aldolase (43,000), carbonic liant Blue. The proteins used as molecular weight markers were

anhydrase (30,000), soybean trypsin inhibitor (20,100), and a-lactal-

bonyl; MCA, methylcoumarylamide; NA, 2-naphthylamide; MNA, 4- ' The abbreviations used are: SUC-, succinyl; Cbz, N-benzyloxycar-

methoxy-2-naphthylamide; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; BSA, bovine serum albumin; FPLC, fast protein liquid chromatography.

bumin (14,400). Various acetylated cytochromes c with PI values of 4.1,4.9,6.4,8.3,9.7, and 10.6 were used as PI markers. Reverse-phase HPLC was performed as follows: Samples of 0.5 mg of purified enzymes were applied directly to a Cosmosil 5C4-300 column (10 X 250 mm) equilibrated with 0.05% trifluoroacetic acid. Gradient elu- tion was performed with an increasing concentration of acetonitrile containing 0.05% trifluoroacetic acid in a Waters model 141 HPLC system at a flow rate of 1 ml/min. The elution was monitored as absorbance at 280 and 215 nm. Amino acid analysis was performed as described previously (2). The amount of cysteic acid was not determined.

Physicochemical Analyses-Analytical ultracentrifugation and measurements of quasi-elastic light scattering and circular dichroism were performed as described previously (2).

Electron Microscopy-Details of the method were described else- where (19). A sample of about 100 pg of purified enzyme was applied to a TSK G4000SW column (Toyo Soda, Tokyo) connected to an HPLC system (Jasco, Tokyo) and materials were eluted with 100 mM ammonium acetate buffer (pH 7.4). The elution was monitored by measuring the absorbance at 280 nm. Fractions in the major peak containing monomeric forms of the enzyme were collected for electron microscopic examination. The enzyme was negatively stained on a glow-discharged, carbon-coated Formvar membrane with 3% uranyl acetate at pH 4.5. Negatively stained samples were examined in a Hitachi H7000 electron microscope at a direct magnification of X 50,000.

Immunological Analysis-Antisera against various proteasomes were raised in rabbits, and their IgG fractions were prepared as described previously (I). The standard procedure was used for Ouch- terlony double-diffusion tests (42). Immunoelectrophoretic blot analysis was performed by a modification of the method of Towbin et al. (43). Briefly, samples (25 pg of protein) separated by SDS- PAGE in 12.5% gel were transferred electrophoretically to Durapore membranes (Millipore) with a semi-dry electroblotter (Sartorius). The membranes were treated with 3% BSA and then with various anti-proteasome antibodies (10 pglml) and '"I-protein A (0.25 pCi/ ml). They were then washed extensively with 0.05% Tween 20 solu- tion and autoradiographed at -70 "C.

method of Bradford (44) with BSA as a standard. The protein Protein Assay-Protein concentration was measured by the

concentration of the purified enzyme was calculated from the absorb- ance at 280 nm, assuming E!:m values of 11.2 (human liver), 9.6 (rat liver), 10.8 (chicken liver), 12.3 (X. lueuis ovary) and 7.4 (yeast proteasomes). These values were determined with a refractometer as described by Lodola et at. (45) using a solution of BSA of known absorbance as a standard.

RESULTS

I. Functional Properties of Proteasomes

Latency-We have reported that a high molecular weight protease from rat liver is present in a latent state in cells and can be activated in various ways (1). Therefore, we examined whether the enzymes from other species also have latent proteinase activity. Table I shows that all the purified en- zymes have very low activity for the breakdown of [3H]casein, but that their activities are strongly activated by addition of poly-L-lysine, the most effective activator of the rat liver enzyme. These enzymes were also activated in artificial ways such as by heat treatment and trypsin digestion, as reported previously for the enzyme from rat liver (data not shown). Thus, these results clearly indicate that all these enzymes show latent activity for protein hydrolysis.

Substrate Specificity-The activities of the enzymes from five different species on low molecular weight peptides were examined. The enzymes degraded three types of fluorogenic oligopeptides with acidic, basic, and neutral hydrophobic amino acids at their carboxyl termini; namely, Cbz-Leu-Leu- Glu-NA, Cbz-Ala-Arg-Arg-MNA, and Suc-Leu-Leu-Val-Tyr- MCA. Fig. 1 shows the pH dependence of their activities on these typical substrates. The pH optima for Suc-Leu-Leu- Val-Tyr-MCA and Cbz-Leu-Leu-Glu-NA were in the range of 8.0-8.5, whereas those for Cbz-Ala-Arg-Arg-MNA were in

The Proteasomes (Eukuaryotic Multi-protease Complexes) TABLE I

Latent proteolytic activity of purified proteasoms Purified enzymes (5 pg of protein) were used for assays. Poly-L-lysine (34 kDa) was added at a final concentration

of 0.5 mdml. Data are means f S.D. for three different exDeriments.

16211

Degrading activity of [methyl-3H]casein Addition Human Rat Chicken x. loeuis

liver liver liver ovarv Yeast

%/h None 1.9 f 0.3 3.8 f 0.5 2.5 f 0.4 6.6 f 0.5 8.2 f 0.6 Poly-L-lysine 18.4 f 2.2 23.1 f 3.6 18.0 k 4.0 34.7 f 5.9 35.4 f 5.2

PH FIG. 1. Effect of pH on the activities of various enzymes on

the three fluorogenic substrates. Tris-HC1 (0.1 M) was used, and the pH of the assay mixture was measured directly after increasing the volume to 1 ml. SDS at a final concentration of 0.05% was added to the assay mixtures with Suc-Leu-Leul-Val-Tyr-MCA (left panel) and Cbz-Leu-Leu-Glu-NA (right panel), but not with Cbz-Ala-Arg- Arg-MNA (middle panel). Values for human (O), rat (O), chicken (A), Xenopus (V), and yeast (X ) proteasomes are shown as percent- ages of the maximum activity.

the range of 9.0-9.5. The pH dependence of the cleavage reactions of these three peptides were very similar for all enzymes, suggesting that all eukaryotic enzymes can be re- garded as functionally similar types of proteases. Interest- ingly, the activities for degrading Cbz-Leu-Leu-Glu-NA and Suc-Leu-Leu-Val-Tyr-MCA were markedly increased by 0.02-0.05% SDS, but the degrees of stimulation of different preparations of the enzymes varied greatly (3-30-fold). In contrast, the hydrolytic activity for Cbz-Ala-Arg-Arg-MNA was partially reduced by the addition of a low concentration of SDS (data not shown).

Inhibitor Sensitivity-We then examined the effects of various inhibitors on the activities on the three substrates. The inhibitor spectra of the enzymes from all sources were similar (data not shown). Leupeptin and chymostatin prefer- entially inhibited the cleavages of Cbz-Ala-Arg-Arg-MNA and Suc-Leu-Leu-Val-Tyr-MCA, respectively. In contrast, these two inhibitors had no effect on the hydrolysis of Cbz-Leu- Leu-Glu-NA. These three activities of all enzymes were in- hibited, although to different extents, by sulfhydryl-blocking agents, such as N-ethylmaleimide and 5,5'-dithiobis-(2-nitro- benzoate), but Ep-475, a specific cysteine-protease inhibitor, had no effect on any of the activities. Therefore, the enzymes cannot be considered to be thiol proteases, although thiol residues appear to play a critical role in their activities. Phenylmethylsulfonyl fluoride and diisopropyl fluorophos- phate inhibited the three hydrolytic reactions to different extents. Since these compounds are covalent modifiers of serine residues of enzyme proteins, the enzymes are thought to be serine proteases. In addition, hemin had different effects on these three activities. These findings strongly suggest that the hydrolyses of these three peptides are catalyzed at distinct catalytic sites, as suggested previously (1,8, 11). Thus, all the data are compatible with the idea that the enzymes all have multiple catalytic sites within a single protein complex.

II. Structural Properties of Proteasomes Physicochemical Properties-First, we examined the phys-

icochemical properties of proteasomes purified from various cells and tissues. The results are summarized in Table 11. All the proteasomes sedimented as single components with sedi- mentation coefficients (szo,,,,) of 19 S to 22 S. Measurements by quasi-elastic light scattering gave diffusion coefficients (Dz0,,,,) of 2.02-2.60 X cm2 s" for these enzymes. Their effective hydrodynamic (Stokes) radii were calculated to be 82-106 A according to the Einstein-Stokes relation. From the values of s20,,,, and Dz0,,,,, the molecular masses of these enzymes were estimated to be 710-910 kDa by Svedberg's equation. These results indicate that all the enzymes in species ranging from man to yeast have a similar large size. The similarity in size of these proteasomes was supported by the observation that all the enzymes had almost the same retention time on molecular sieve chromatography on FPLC on a Superose 6 column (data not shown). The PI value of the yeast enzyme was estimated to be 4.6 and those of the native enzymes of human, rat, and chicken liver, and X. h v i s were found to be approximately 5.0 by isoelectric focusing.

Morphology-We have reported that rat liver proteasomes appear byoelectron microscopy to be Fing-shaped particles of 160 f 10 A in diameter $nd 110 f 7 A in height with a small central hollow of 10-30 A in diameter (19). The enzyme from rat skeletal muscle was reported to have similar morphology (18). Here, we examined the morphology of proteasomes from other eukaryotic cells. Fig. 2 shows electron micrographs of the enzymes purified from human, chicken, Xenopus, and yeast after negative staining with uranyl acetate. Interest- ingly, all these enzymes showed very similar morphology, appearing as ring-shaped particles with a well-defined symmetrical structure and a small hole in the center. The particle diameters of proteasomes of four different species ( N = 100) were measured at higher magnification (Fig. 2, right- hundphotogruphs). The diameters (average f S.D.) of human, chicken, and yeast pcoteasomes were, respectively, 127 f 7, 124 k 7, and 123 f 7 A, whereas that of Xenopus proteasomes was significantly larger, being 153 f 12 A, with a broader distribution of values, like that of rat liver proteasomes. Since these proteasomes showed no difference in either molecular weight or Stokes radius, as described in the previous section, the difference in the average diameter seen by electron mi- croscopy could be due to distortion of molecular shape during preparation of rat and Xenopus proteasomes.

The internal structure of proteasomes is difficult to study due to complexity of subunit proteins. But two interesting observations were made. First, in many cases, particularly in human proteasomes, we detected semiglobular units of ap- proximately 30-50 A in diameter along the central hole (Fig. 2A, arrows). These units were too large to be single subunit proteins and probably consisted of several proteins. Second, in about one-fourth to one-third of the proteasomes of all four species, one or two slits connecting the central hole to the

16212 The Proteasomes (Eukaryotic Multi-protease Complexes)

TABLE I1 Physicochemical properties of proteasomes

Values of S Z O , ~ , Dzo,w, and PI were measured as described under "Experimental Procedures" and in Ref. 2. Values for molecular weight were estimated from values of s ~ , ~ , and those of D20,u, using the Svedberg equation. Stokes radii were calculated from values of D z o , ~ . Values of molecular ellipiticity ([6']zz0.,, based on residue molarity) were calculated from circular dichroism measurements.

Enzyme source SZ0.U D20.l" M, Stokes radius PI [&O"rn

s ern2 s"

Human liver 21.8 2.28 X Rat liver" 19.8 2.50 X 10" Chicken liver 20.0 2.02 x 10" X . laevis ovary 19.6 2.15 X Yeast (S. cereuisiae) 20.0 2.60 X

Data are from Tanaka et al. (2).

outside were clearly visible (Fig. 2, B and D, and arrows in D). These observations suggested that the proteasomes are rather fragile or flexible molecules consisting of 6-8 substruc- tures, each containing several subunit proteins.

Subunit Structure-We then examined the subunit struc- tures of these proteasomes. When the purified enzymes were denatured with 1% SDS and subjected to electrophoresis on polyacrylamide gel with 0.1% SDS, they gave multiple bands in the range of 22-33 kDa (Fig. 3). The patterns were similar in the presence and absence of a sulfhydryl-reducing reagent (data not shown). Although the enzymes from different spe- cies were all separated into smaller polypeptides with a similar molecular weight range, the exact electrophoretic mobilities or sizes of the resulting polypeptides were somewhat different. Moreover, on isoelectric focusing after dissociation with 8 M urea, these enzymes showed strikingly different PI values of 3-11, and clear species differences in patterns (PI variations) (Fig. 4). Moreover, when these multiple components were separated by reverse-phase HPLC on a Cosmosil 5C4-300 column (Fig. 5), they showed different elution profiles: the profiles of those from Xenopus laevis and yeast were especially different from those of the other three species. These differ- ences indicate that proteasomes from different sources have minor interspecies differences in subunit multiplicity.

Spectral Properties-The absorption spectra of these five proteasomes resembled each other and exhibited a maximum at 278 nm with a small shoulder at 283 nm (data not shown). The extinction coefficients, at 280 nm were calculated to be 11.2 (human liver), 9.6 (rat liver), 10.8 (chicken liver), 12.3 (X. laevis ovary), and 7.4 (yeast). No peak or shoulder was detected at around 260 nm in any preparation, indicating that they were all free from nucleic acids.

The circular dichroism spectra in the far-ultraviolet region for all proteasomes showed the double minimum characteris- tics of an cy-helical structure (Fig. 6). The spectrum of the rat liver enzyme has been reported (2). The minimum values of the mean residual ellipticity at 220 nm were calculated to be -9,800 (human), -12,000 (rat), -10,300 (chicken), -11,600 (Xenopus), and -9,600 (yeast). The similar patterns of these CD spectra suggest that all these proteasomes are similar in secondary structure.

Amino Acid Composition-The amino acid compositions of the five proteasomes are shown in Table 111. All the enzymes except the yeast enzyme had remarkably similar amino acid compositions. The yeast enzyme differed in certain amino acids such as Asx, Arg, Ile, Phe, Tyr, Met, and Pro. The higher content of Asx and lower content of Arg of the yeast enzyme may account for its acidic nature shown by its PI value (Table I).

A cm2 mol" degrees

870,000 94 5.0 -9,000 720,000 85 5.0 910,000

-12,000 106

840,000 98 4.9 -10,300 5.0 -11,600

710,000 82 4.6 -9,600

III. Immunological Properties of Proteasomes

We next examined the immunological reactivities of these proteasomes. Polyclonal antibodies against the enzymes from the five species were raised in rabbits. As shown in Fig. 7, the enzymes were all clearly distinguishable by the Ouchterlony double-diffusion test, indicating species-specific differences in immunological reactivity. Previously, we found that the an- tibody against the rat liver enzyme cross-reacted with en- zymes from other tissues, such as skeletal muscle, kidney, heart and brain of the same rat (I), indicating the absence of tissue specificity of proteasomes in the same species. The species-specific immunological differences seem to have been acquired during evolution.

We then examined whether the species-specific differences revealed by Ouchterlony analysis were due to differences in immunoreactivity of subunit components of the various pro- teasomes. Fig. 8 shows the results of immunoelectrophoretic blot analyses. In all cases, the antibody against one enzyme reacted with almost all the subunits of that enzyme, although the reactivities with individual components were not corre- lated with their protein contents. Moreover, the antibody against an enzyme of one species also partially cross-reacted with some components of the enzymes of other species: the antibody against the enzyme from rat liver reacted strongly with one or two subunits of other enzymes (Fig. 8C), whereas the antibodies to the enzymes from human, chicken, X. laevis, and yeast reacted weakly, but significantly, with various com- ponents of other enzymes (Fig. 8, B, D, E, and F). These partial cross-immunoreactivities with subunit components of other enzymes observed under denatured conditions using polyclonal antibodies against native enzymes were not con- sistent with the results of Ouchterlony immunodiffusion analysis (Fig. 7). These differences can be explained by sup- posing that some subunits in each enzyme complex show the same immunoreactivity, but that these subunits are located in the complexes in such a way as not to react with antibodies. The existence of several components with similar immuno- reactivities in different species suggests that some subunits, or domains, of the various proteasomes have been conserved during evolution.

DISCUSSION

Physiological Role of Ubiquitously Distributed Protea- somes-In the present study, we purified proteasomes from cells of a variety of eukaryotes ranging from man to yeast. All the eukaryotic proteasomes obtained by the present study had the same enzymological function. The enzymes had two unique properties: latency of proteolytic function and multi- plicity of catalytic sites within a single molecular complex, as

The Proteasomes (Eukaryotic Multi-protease Complexes) 16213

1 2 3 4 5

. ., :.?

I

f

.”

- - FIG. 2. Electron micrographs of proteasomes. Source of pro-

teasomes: A, human liver; I?, chicken liver; C, X . [aeois ovary; D, yeast. Bars on. the left represent 1000 A and those on the right represent 200 A. Samples were negatively stained with 3% uranyl acetate. Photographs a t right show the similarity of different particles selected a t random. Arrows in A and R-D indicate semiglobular units and slits, respectively (see text).

in rat liver proteasomes (1). Their molecular structures may be responsible for this latency, since some conformational change could easily affect the catalytic activity of the complex. The latency may be important physiologically for regulation of intracellular proteolysis. The multicatalytic activities may

20” 0 m

14- - - ”” -

FIG. 3. SDS-PAGE analysis of proteasomes from various eukaryotes. SDS-PAGE was carried out as described under “Exper- imental Procedures.” Lanes 1-5 show the electrophoretic patterns of the enzymes from hnuman, rat, and chicken liver, X . laeuis ovary, and yeast, respectively. The left and right lanes are M, marker proteins. Samples of 25 pg of the enzymes were used. Proteins were stained with Coomassie Brilliant Blue.

1 2 3 4 5

8.3- ;

9.7- i

10.6- , .

FIG. 4. Urea-isoelectric focusing analysis of proteasomes from various eukaryotes. Urea-isoelectric focusing was carried out as described under “Experimental Procedures.” Lanes 1-5 show the isoelectric focusing patterns of the enzymes from human, rat, and chicken liver, X . laeuis ovary, and yeast, respectively. Bars on the left show the positions of various acetylated cytochromes c used as PI markers. Samples of 25 pg of the enzymes were used. Proteins were stained with Coomassie Brilliant Blue.

be involved in cascade or cooperative reactions in the prote- olytic pathway, in which they would be advantageous for rapid proteolysis, or they might be responsible for post-translational modification of various proteins, such as in processing of various precursor proteins.

This work showed that proteasomes are present in a wide variety of eukaryotic organisms ranging from man to yeast, implying that these proteasomes are generally important in proteolytic functions. Proteasomes are extralysosomal pro- teases and probably play an essential role in the so-called nonlysosomal proteolytic pathway, which is thought to be responsible for selective degradation of abnormal proteins. In

16214 The Proteasomes (Eukuryotic Multi-protease Complexes)

I I Human Liver

Rat Liver 8 e s c 0

P U Q > 0 a

- c. - a 1

b am &me l a 8 am. 11. zm ’ 8 4 I 1 Chicken Liver

I Xenopus Laevir Ovary

Yeast

Retention Time (min) FIG. 5. Separation of multiple components from proteasomes by reverse-phase HPLC. Chromatogra-

phy on a Cosmosil5C4-300 reverse-phase column was carried out as described under “Experimental Procedures.” Samples of 0.5 mg were used for analyses at 280 nm (-, 0.025 absorbance units) and 215 nm (- - -, 0.25 absorbance units). The blank run, performed by injecting 200 p1 of buffer, indicates that two peaks with retention times of about 25 min and 300 min are artifacts due to injection of samples and increase in the acetonitrile concentration, respectively.

all living cells of prokaryotes and eukaryotes, rapid proteolysis is important for preventing the accumulation of unnecessary proteins, which are continuously generated in cells by muta- tion of genes, errors of transcription and translation, and postsynthetic damage of various proteins (46). The wide dis- tribution of proteasomes is consistent with these ubiquitous cellular functions. But these enzymes do not appear to be present in bacteria such as Escherichia coli, suggesting that proteasomes are present only in eukaryotic cells. Interest- ingly, we recently found that proteasomes are partly localized in the nucleus and that their concentration is high in mam- malian liver: suggesting their biological importance for nu- clear proteolysis. This may be another reason for the presence of proteasomes in only eukaryotic cells.

K. Tanaka, et al., manuscript submitted for publication.

Similarity and Interspecies Structural Variation of Various Proteasomes-In the present study, we also showed that pro- teasomes resembled each other in gross structure. They all had unusually large molecular masses of 700-900 kDa and sedimentation values of approximately 19 S to 22 S (Table 11), and appeared to be symmetrical ring-shaped particles by electron microscopy (Fig. 2). In addition, they had similar amino acid compositions (Table 111) and secondary structures, as judged by their circular dichroic spectra (Fig. 6). Thus, the gross structures of proteasomes appear to have been highly conserved during evolution of eukaryotes.

Interestingly, all the proteasomes were multisubunit com- plexes consisting of 15-20 small polypeptides with similar molecular masses of 22-23 kDa (Fig. 3), but with strikingly different PI values of 3-10 (Fig. 4). Previously, we reported that multiple components isolated from rat liver proteasomes

The Proteasomes (Eukaryotic Multi-protease Complexes) 16215

Wavelength (nrn)

FIG. 6. Circular dichroism spectra in the far-ultraviolet re- gion. Spectra were obtained at protein concentrations of 0.9-1.5 mg/ ml. Results are averages of 16 accumulations of the enzyme from human (A) , chicken ( B ) , Xenopus ( C ) , and yeast (D) in solution containing 25 mM Tris-HC1 (pH 7.5), 0.1 M KC1, and 0.2 mM dithio- threitol. A cell with a light path of 0.1 cm was used.

TABLE 111 Amino acid compositions of the proteasomes

Human Rat Chicken X. loeuis Yeast

mol %

liver livef liver ovary

Asparagine or 8.4 8.7 8.2 8.7 11.0

Glutamine or 12.4 11.2 13.1 12.5 12.9

Arginine 5.5 5.4 5.4 5.2 3.6 Lysine 6.2 5.5 6.0 6.7 6.2 Histidine 1.9 1.9 1.9 1.8 1.3 Alanine 9.5 9.0 9.4 9.5 8.9 Glycine 8.1 8.0 8.5 7.9 9.4 Leucine 8.8 8.4 8.3 8.7 8.3 Isoleucine 5.7 5.3 5.7 5.8 6.5 Valine 7.2 7.2 7.0 7.1 7.1 Phenylalanine 3.4 3.5 3.3 3.4 2.8 Tyrosine 4.6 4.5 4.5 4.9 3.5 Tryptophan 0.3 0.4 0.4 0.3 0.4 Serine 5.8 6.8 6.1 5.7 6.2 Threonine 5.4 5.8 5.5 5.5 5.5 Half-cystine NDb 1.4 NDb NDb NDb Methionine 3.1 3.1 2.8 3.0 1.8 Proline 3.6 3.0 3.6 3.3 4.5

aspartic acid

glutamic acid

Data from Tanaka et al. (2). ND. not determined.

are nonidentical, as judged by peptide mapping and cell-free translation of mRNA (19). Thus, other proteasomes are prob- ably also composed of multiple, nonidentical subunits. The multiplicity of subunits of similar size but with widely differ- ent PI values was a common feature of the proteasomes, but the subunits showed species-specific differences (Figs. 3-5). Moreover, purified proteasomes of various species did not cross-react on Ouchterlony immunodiffusion analysis (Fig. 7), showing that the immunoreactivities of the proteasomes were species-specific. However, some components of proteasomes showed partial cross-reactivity on immunoblotting analyses

(Fig. 8), suggesting that during evolution some subunits, or domains, of proteasomes have been conserved, although not completely, because cross-reactivity was observed between components with different molecular weights. In addition, the finding that the cross-reactivity of proteasomes of one species with antibody to proteasomes of another species was different from the reverse cross-reactivity suggested that the localiza- tions of antigenic domains, that is, the subunit organizations, in various proteasomes are different.

The resemblance of the gross structures of different protea- somes suggests a common mechanism of their subunit orga- nization. The similarity in subunit size may be important for construction of a symmetrical complex, and the heterogeneity of the net charges of the subunits is presumably related to the assembly of the large multisubunit complex by complemen- tary charge interaction. Thus, the size similarity and charge variation in the subunits may be important for conservation of the proteasomes during evolution, even though the individ- ual components are altered. For further elucidation of the species-specificity in subunit multiplicity of proteasomes, more detailed studies, such as analyses of their gene struc- tures, are required.

Identity of Proteasomes with Ring-shaped 20 S Particles Found in Various Eukaryotic Cells-Proteasomes are struc- turally unique in size, shape, and subunit multiplicity. Inter- estingly, these multi-protease complexes are quite similar to the subcellular 19 S to 22 S particles found during the past 15 years in a wide variety of eukaryotic cells, such as plants (32,38), Drosophila (24-27), Xenopus (28,29), seaurchin (30), birds (31, 38), and various mammals (31-38). So far it has been very difficult to determine their similarity or identity, since they have been isolated from different cells and tissues and in different ways, and the biological functions of most of these particles are unknown.

Recently, we found that human prosomes are identical to proteasomes, judging from their immunological cross-reactiv- ity, morphological appearance by electron microscopy, poly- peptide composition, and proteolytic activities (20). Falken- burg et al. (39) also reported the identity of Drosophila 19 S ribonucleoproteins particles with rat muscle multicatalytic proteinase. In the present work, we demonstrated that pro- teasomes were distributed in a variety of eukaryotic cells. This finding is consistent with the observation of the ubiquitous

these particles are thought to be identical to proteasomes. However, there are several reports of similarity in morphology but differences in homology of these particles (25, 29, 31); namely differences in subunit composition and immunological reactivity of apparently similar particles. Therefore, it has sometimes been claimed that these particles are closely re- lated, but not identical. In the present work, we found that proteasomes from a variety of eukaryotic cells exhibit inter- species variations in subunit compositions (Figs. 3-5) and immunological reactivities (Figs. 7 and 8), suggesting that the apparently discrepant results reported previously are attrib- utable to minor structural variations of homologous mole- cules, which may be acquired during evolution. Thus all the 20 S particles described previously can be regarded as protea- somes showing minor differences.

There are, however, major differences between the ring- shaped particles reported in the literature and those observed in this work. Prosomes and 19 S ring-type particles containing heat-shock proteins have been considered to be ribonucleo- protein particles that have significant amounts (15-20% of the total molecular weight) of specific RNA species (25, 27, 31, 38). In contrast, the proteasomes demonstrated by us (2,

&-t 13 rlbution . of these 19 S to 20 S particles (31, 38). Thus, all

16216 The Proteasones (Eukaryotic Multi-protease Complexes)

Human Liver Rat Liver Chicken Liver Xenopus Laevis Ovary Yeast

1 1 1

4 4. 4 4 4 - ., FIG. 7. Ouchterlony double-diffusion analysis of proteasomes from various eukaryotes. Samples of

10 pg of the enzymes from the various sources indicated at the top of each panel were placed in the center well ( E ) . Anti-serum (7 pl) to samples from human liver ( I ) , rat liver (2), chicken liver (3), X. laevis ovary (4), and yeast (5) and nonimmunized control serum (6) were placed in the side wells. Samples were incubated for 2 days a t room temperature in a humidified chamber, washed for 4 days with phosphate-buffered saline with gentle shaking, and then stained with Coomassie Brilliant Blue.

1 2 3 4 5

B

D

E

F

FIG. 8. Immunoelectrophoretic blot analysis of various pro- teasomes. Immunoblotting analysis was performed as described un- der “Experimental Procedures.” Lanes 1-5 show the electrophoretic patterns of the enzymes from human, rat, and chicken liver, X. luevis ovary, and yeast, respectively. Panel A shows the patterns of various proteasomes stained with Coomassie Brilliant Blue. Panels B-F show the patterns of various proteasomes after immunostaining with an- tibodies against the enzymes from human, rat, and chicken liver, X. laevis ovary, and yeast, respectively.

19, and this paper) and 5’-pre-tRNAase (29) and 22 S particles from X. laeuis (28) did not contain any RNA. These differ- ences seem to be due to differences in the procedures used for purification of the particles: particles containing small RNA moieties have been isolated by the mild procedure of sucrose density gradient centrifugation in the presence of a high salt

concentration to prevent artificial adsorption of RNA con- taminants, whereas particles without RNAs have been puri- fied by more drastic ion-exchange and hydroxylapatite chro- matographic techniques. Thus particles per se may consist of a core part, in which the subunits are very tightly assembled, and additional components that bind loosely to the core part and are probably lost during drastic purification procedures. Functionally, the core part may be a multi-enzyme complex containing protease and probably nuclease, and the additional part may contain some factors such as small RNA moieties and/or certain heat-shock proteins, which presumably play a regulatory role in the enzymatic functions of the core part. Thus, the subcellular 20 S particles in a variety of eukaryotic cells must be homologous.

In this paper, we describe proteasomes only as multi-pro- tease complexes; that is, particles with protease activities. However, these enzymes may also have other as yet unknown functions. Such functions are suggested from the structural similarity of proteasomes to many kinds of 20 S particles. For example, some functions of 20 S ring-shaped particles have been reported. The ribonucleoproten particles named “pro- somes,” have been characterized and are believed to function in the regulation of translation, because they are co-purified with the inactive mRNA complex (24) and because their RNA moiety has a common complementary sequence in different mRNAs (31). In addition, in Drosophila, 19 S ring-shaped particles are tightly associated with polyribosomes (27). More- over, 19 S cytoplasmic particles have been shown to have small heat-shock proteins and to be identical to the prosomes in Drosophila (25,26). Furthermore, Castano et al. (29) found that similar particles are co-purified as a pre-tRNA processing nuclease in X. lueuis. These results suggest that the 20 S particles contain small RNA species and that certain heat- shock proteins may function in RNA processing, in the reg- ulation of protein synthesis, and in the response of eukaryotic cells to environmental stress; in other words, proteasomes may be multifunctional enzyme complexes with both protease and nuclease activities and may function in both RNA and protein metabolism in all eukaryotic cells. The restriction of many biologically essential functions to one particle may be very efficient for cellular activities.

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