Components of Ubiquitin-Protein Ligase System

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 258, No. 13, Issue of July 10, pp , 1983 Printed In U.S.A Components of Ubiquitin-Protein Ligase System RESOLUTION, AFFINITY PURIFICATION, AND ROLE IN PROTEIN BREAKDOWN* (Received for publication, December 27, 1982) Avram HershkoS, Hannah Heller, Sarah Elias, and Aaron Ciechanover From the Unit of Biochemistry, Faculty of Medicine, Technwn-Israel Institute of Technology, Haifa, Israel By affinity chromatography of a crude reticulocyte extract on ubiquitin-sepharose, three enzymes required for the conjugation of ubiquitin with proteins have been isolated. One is the ubiquitin-activating enzyme (E,), which is covalently linked to the affinity column in the presence of ATP and can be specifically eluted with AMP and pyrophosphate (Ciechanover, A., Elias, S., Heller, H., and Hershko, A. (1982) J. Biol. Chem. 257, ). A second enzyme, designated Ez, is bound to the ubiquitin column when E, and ATP are present, and is eluted with a thiol compound at high concentration. The third enzyme, designated E3, is adsorbed to the affinity column by noncovalent interactions and can be eluted with high or increased salt ph. The presence of all three enzymes is absolutely required for the conjugation of '251-~biq~itin with proteins. All three affinity-purified enzymes are also required for the breakdown of 1251-albumin to acid-soluble material in the presence of ubiquitin, ATP, and the unadsorbed fraction of the affinity column. jugation and protein breakdown was corroborated by experiments with intact cells, in which striking correlations were observed between the rapid degradation of abnormal proteins The following observations indicate that the function and increased formation of ubiquitin-protein conjugates (10, of E2 is the transfer of activated ubiquitin to the site of 11). conjugation in the form of an Ez-ubiquitin thiol ester Our knowledge of the intermediary steps in the ATPintermediate. (a) E2 is rapidly inactivated by iodoacet- ubiquitin proteolytic pathway is still rudimentary. The conamide, but can be protected against inactivation by a jugation of ubiquitin with proteins is apparently initiated by prior incubation with El, ATP, and ubiquitin. This suggests an El-mediated transfer of activated ubiquitin to an iodoacetamide-sensitive thiol site of E2. (b) The requirements for the binding of Ez to the ubiquitin column and the mode of its elution, cited above, are consistent with the notion that a covalent linkage is formed between E, and Sepharose-bound ubiquitin. (c) Upon the incubation of '251-ubiquitin with E, and ATP, followed by the addition of purified E,, activated ubiquitin is transferred from El to several low molecular weight forms of Ez, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The linkage of ubiquitin to all these forms has the characteristics of a thiol ester bond. In a further incubation with E3 and a protein substrate for conjugation, activated ubiquitin was transferred from the different forms of Ez-ubiquitin to stable ubiquitin-protein conjugates. Thus, E3 is involved in the last step of the ligase system. * This work was supported by United States Public Health Service Grant AM and funds from the Institute for Cancer Research to Irwin A. Rose. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported by United States Public Health Service Grant AM and a grant from the United States-Israel Binational Science Foundation. The energy dependence of the degradation of intracellular proteins has been recognized a long time (1, 2), but its mechanisms remained unknown. Recent studies on the mode of action of an ATP-dependent proteolytic system for reticulocytes led to the identification of a pathway of protein breakdown. The system is composed of several essentially required components (3), including a small, heat-stable polypeptide (4, 5). The polypeptide was subsequently identified as ubiquitin, a universally occurring protein of previously unknown func- tion (6). Ubiquitin is covalently bound to reticulocyte proteins or exogenous protein substrates in an ATP-requiring process, in which several molecules of the polypeptide are conjugated to the substrate protein by amide linkages (7,8). A model was proposed according to which the conjugation of ubiquitin with proteins is the initial signal event in protein breakdown (reviewed in Ref. 9). The relationship between ubiquitin con- a specific ubiquitin-activating enzyme, first identified by ubiquitin-dependent PP,:ATP and AMP:ATP exchange reactions, and by the binding of activated ubiquitin to enzyme in a thiol ester linkage (12). A mechanism involving the formation of ubiquitin adenylate and its transfer to a thiol site of the enzyme was proposed (12) and substantiated by direct evidence (13, 14). Ubiquitin is activated at its COOHterminal glycine (15), which is in accord with the observation that this residue is bound by isopeptide linkage to enh2 lysine in a ubiquitin-histone conjugate (16). The ubiquitin-activating enzyme was purified to near homogeneity by a covalent affinity chromatography procedure, in which the enzyme is first bound to ubiquitin-sepharose in the presence of ATP as a thiol ester intermediate, and is then specifically eluted with AMP and pyrophosphate (17). The activating enzyme does not form ubiquitin-protein conjugates by itself (12, 17), but it is a donor for conjugate formation in the presence of a crude reticulocyte extract (13). In the present report, we describe two further enzymes which participate in the conjugation of ubiquitin with proteins. Evidence is presented which indicates the involvement of the three components of the ubiquitin-protein ligase system in protein degradation. MATERIALS AND METHODS Ubiquitin was purified from human erythrocytes by a modification (17) of a previously described method (5). Ubiquitin and bovine serum albumin (Pentex) were radiolabeled with NalWI (Nuclear Research Center, Negev, Israel) by the chloramine-t method, as described (7). 8206

2 Ubiquitin-Protein Ligase System 8207 RNase A (bovine pancreas, type I-A) was purchased from Sigma and was subjected to performic acid oxidation as described by Hirs (18). Creatine phosphokinase (150 units/mg) and yeast inorganic pyrophosphatase (550 units/mg) were purchased from Sigma, and yeast hexokinase (140 units/mg) from Boehringer Mannheim. Crystallized hen ovalbumin was obtained from Worthington. Preparation of Reticulocyte Fractions-Reticulocyte extracts were prepared by a modification of previously described procedures (3,4). Briefly, reticulocyte-rich blood (70-90% reticulocytes) was obtained from rabbits following injections of phenylhydrazine (19). The cells were washed twice with phosphate-buffered saline (150 mm NaC1, 10 mm potassium phosphate (ph 7.4)), suspended in an equal volume of Krebs-Ringer phosphate medium lacking glucose (19), and incubated at 37 "C for 90 min with 0.2 mm 2,4-dinitrophenol and 20 mm 2- deoxyglucose. This treatment of ATP depletion is required to release ubiquitin from endogenous ubiquitin-protein conjugates. All subsequent operations were carried out at 0-4 "C. The cells were washed twice with phosphate-buffered saline, lysed with 1.5 volumes of 1 mm dithiothreitol, and centrifuged at 80,000 X g for 90 min to remove particulate material. Crude lysates could be stored at -80 'C for over a year without loss of activity. Lysates were fractionated on a column of DEAE-cellulose (Whatman DE52) equilibrated with 3 mm potassium phosphate (ph 7.0) and 1 mm dithiothreitol, at a ratio of lysate to resin of 1.5:l (by volume). Unadsorbed material (Fraction I, containing ubiquitin (4)) was collected and the column was washed with 2.5 column volumes of a buffer containing 3 mm potassium phosphate (ph 7.0), 1 mm dithiothreitol, and 20 mm KCl. Proteins adsorbed to the resin (Fraction 11) were eluted with 2.5 column volumes of a solution consisting of 0.5 mm KCl, 20 mm Tris-HC1 (ph 7.2), and 1 mm dithiothreitol. The eluate was concentrated by ammonium sulfate precipitation and dialyzed as described (4). Following dialysis, some insoluble material was removed by centrifugation (20,000 X g, 15 min). The final volume of Fraction I1 was usually one-fifth of the starting volume of crude lysate, and its protein concentration was in the range of mg/ ml. Fraction I1 contains all the enzymes required for ATP-uhiquitindependent protein breakdown (3,4) and for the formation and breakdown of uhiquitin-protein conjugates (7,8). It can be stored at -80 "C for at least a year without loss of proteolytic activity, provided that ATP (0.5 mm) is added to protect an ATP-stabilized factor (3). Without ATP, Fraction I1 was stored at -80 "C in small samples, thawed only once, and used within 2-3 weeks of preparation. Affinity Chromatography-Affinity chromatography of the components of the ubiquitin-protein ligase system was performed by an extension of the procedure described previously for the purification of ubiquitin-activating enzyme (17). Ubiquitin was coupled to activated CH-Sepharose (Pharmacia Fine Chemicals, Piscataway, NJ) as described previously (17), except that the concentration of Sepharose-bound ubiquitin was approximately 20 mg/ml of swollen gel. This high concentration of Sepharose-bound ubiquitin was required for efficient binding of E3 (see "Results"), whereas El and E, were completely hound to columns containing much less ubiquitin (around 5 mg/ml of gel). Column operations were performed at room temperature, but enzyme fractions were collected on ice. A 6-ml column of ubiquitin-sepharose was equilibrated with 5 column volumes of a buffer consisting of 50 mm Tris-HC1 (ph 7.2), 2 mm ATP, 5 mm MgClz, and 0.2 mm dithiothreitol (Buffer A). Fraction I1 from reticulocytes (6 ml) was adjusted to 50 mm Tris-HC1 (ph 7.2), 5 mm ATP, 10 mm MgCI,, and 0.2 mm dithiothreitol and applied to the column at a flow rate of 0.5 ml/min. The unadsorbed fraction was collected until the end of the yellowish concentrated protein color; the protein concentration of the unadsorbed fraction was diluted about 1.5-fold relative to that of Fraction 11. The column was washed with 3 column volumes of Buffer A and then sequentially eluted with the following solutions: 1 M KC1 containing 50 mm Tris-HCI, ph 7.2 (KC1 eluate); the above Tris buffer, to remove salt; 2 mm AMP and 0.04 mm sodium pyrophosphate in the above Tris buffer (AMP-PPi eluate), to elute ubiquitin-activating enzyme (17); 10 mm dithiothreitol in the same Tris buffer at ph 7.2 (DTT' eluate); and 50 mm Tris-HCI (ph 9.0) containing 2 mm dithiothreitol (ph 9 eluate). Each elution was with 3 column volumes of the respective buffer, except for the KC1 elution which was with 6 column volumes. The ph 9 eluate was neutralized with 100 mm Tris-HCI (ph 7.2) immediately following elution. All column eluates were concentrated by centrifuge ultrafiltration with The abbreviations used are: DTT, dithiothreitol; SDS, sodium dodecyl sulfate; E,, ubiquitin-activating enzyme. CF-25 Centriflo membrane cones (Amicon Corp., Lexington, MA), and the buffers were changed by three successive 10-fold dilutions with 20 mm Tris-HCI (ph 7.2) containing 1 mm dithiothreitol, followed by ultrafiltration in the cone. The final volume of the column eluates was brought to 5% of the starting volume of Fraction 11. Protein concentration of the various fractions was determined by the method of Lowry et al. (20). Preparations were stored at -80 "C in small samples. The ubiquitin-sepharose column was regenerated by washing with 10 column volumes of 50 mm Tris-HC1 (ph 9.0) containing 1 M KCl, followed by 10 column volumes of 50 mm Tris-HC1 (ph 7.2) containing 0.02% NaN3. The column was stored in the last buffer at 4 "C and was reused many times over a period of 2 years. The uhiquitin-activating enzyme used in this study was further purified from the AMP-PP, eluate by gel filtration chromatography on a column (0.9 X 60 cm) of Sephacryl S-200 (Pharmacia) equilibrated with 20 mm Tris-HCI (ph 7.2), 1 mm dithiothreitol, and 1 mg/ml of ovalbumin. The peak of enzyme activity (located by the ubiquitin-dependent 3'PPi-ATP exchange assay (17)) was collected and stored at -80 'C in small samples. Ovalbumin was included in the elution buffer for enzyme stabilization, since it is a relatively inert protein for the ubiquitin-atp system. In concentrations up to 2 mg/ ml, ovalbumin does not inhibit the degradation of 1251-albumin, it does not form conjugates with '251-~biq~itin, and 1z51-ovalbumin is not broken down significantly by the reticulocyte proteolytic system (data not shown). Assay of Protein Breakdown-The breakdown of '251-labeled bovine serum albumin ('%I-albumin) to acid-soluble material was determined essentially as described (3). The reaction mixture contained, in a final volume of 50 pl, 50 mm Tris-HC1 (ph 7.6), 5 mm M&~z, 3 mm dithiothreitol, 0.5 mm ATP, 10 mm creatine phosphate, 5 pg of creatine phosphokinase, 4 pg of ubiquitin, 1-2 pg of Iz5I-albumin (5-10 X IO6 cpm), and enzyme fractions as indicated in the legends. Following incubation at 37 "C for 2 h, the reaction was terminated by the addition of 0.8 ml of 5% trichloroacetic acid in the presence of 10 mg of carrier bovine serum albumin. The samples were centrifuged for 3 min in an Eppendorf microcentrifuge, and radioactivity in a 0.5- ml sample of the supernatant was estimated by y counting. Acid- soluble radioactivity present in zero time samples was subtracted, and the results are expressed as the percentage of '251-albumin degraded to acid-soluble material. '2511-albumin was chosen as the substrate for protein breakdown because it is not attacked significantly by non-atp-dependent proteases present in reticulocyte extracts, and thus its degradation is completely dependent upon the supplementation of ATP and ubiquitin (3). Assay of Conjugation of Ubiquitin-Since the determination of the conjugation of '"I-ubiquitin by SDS-polyacrylamide gel electrophoresis (8) is time consuming and laborious, a rapid quantitative assay was developed. The assay is based on the observation that free ubiquitin, which has a neutral isoelectric point, is not adsorbed on either anion or cation exchange resins at neutral ph (7). On the other hand, ubiquitin-protein conjugates are adsorbed on such resins, presumably via their protein moieties. The reaction mixture contained, in a final volume of 50 pl, 50 mm Tris-HC1 (ph 7.2), 2 mm ATP, 5 mm MgClZ, 2 mm dithiothreitol, 0.04 unit of inorganic pyrophosphatase, 20 pg of oxidized RNase, 50 pmol of '9-ubiquitin (about 20,000 cpm), and enzyme preparations as indicated. Oxidized RNase was included since it is a good substrate for conjugation, in contrast to native RNase.' Following incubation at 37 "C for 30 min, the reaction was stopped by the addition of 10 pl of 0.5 N NaOH in the presence of 20 pg of carrier unlabeled ubiquitin. Treatment with alkali was required to release '%I-ubiquitin from thiol ester enzyme intermediates (see"results"), which would be adsorbed to resin in the following step. The addition of carrier ubiquitin was necessary to prevent nonspecific adsorption of tracer amounts of '9-ubiquitin. Following incubation at 37 "C for 5 min, the samples were neutralized by the addition of 10 $1 of 0.5 N HCI. To each sample, 100 p1 of a 50% (v/v) suspension of DE52 and 30 p1 of a similar suspension ofcm52 (Whatman) were added. Both resins had been equilibrated with 10 mm potassium phosphate (ph 7.0) prior to use. The samples were agitated on a Vortex mixer for 10 s, the resins were washed twice with 2-ml portions of 10 mm phosphate buffer (ph 7.0), and resinbound radioactivity was estimated with a y counter. Radioactivity adsorbed to resin in a parallel incubation without enzymes (not more than 10% of total radioactivity) was subtracted, and the results were calculated as picomoles of IZ5I-ubiquitin incorporated into conjugates. A. Hershko and H. Heller, unpublished results.

3 8208 Ubiquitin-Protein Ligase System The assay could be used for estimation of activity of each of the three enzymes of the ligase system (see Results ), provided that the other two were in excess. All assays were performed in the range of linearity with respect to enzyme concentration, which was usually up to 15 pmol of 9-ubiquitin conjugated with proteins. One unit of enzyme activity is defined as the amount of enzyme required for the incorporation of of 251-uhiquitin into conjugates/min under the conditions employed. For the case of the ubiquitin-activating enzyme, this is different from the previously defined unit (17) which was by 32PPi-ATP exchange activity. With the purified activating enzyme, it was estimated that 1 unit of conjugating activity is equivalent to 41 units of PP;-ATP exchange activity. RESULTS Isolation of the Three Factors of the ATP-dependent Proteolytic System by Affinity Chromatography-In a previous study, we have described an affinity procedure for the isolation of ubiquitin-activating enzyme, which was based on the covalent binding of the enzyme to ubiquitin-sepharose in the presence of the proteolytic system are bound to ubiquitin-sepharose. A likely candidate was the ubiquitin-activating enzyme, which is covalently bound to the ubiquitin column and would be of ATP (17). We next examined whether other components removed from Fraction I1 under these conditions. However, of the ATP-ubiquitin proteolytic system can also be isolated the addition of purified ubiquitin-activating enzyme was not by the affinity column. In the experiment. shown in Fig. 1, sufficient to restore protein breakdown in the presence of the reticulocyte Fraction II was applied to ubiquitin-sepharose in unadsorbed fraction and the KC1 eluate (Table I, Experiment the presence or absence of ATP, and the activity of the 1). This suggested the ATP-dependent binding of a further unadsorbed fraction to degrade 1251-albumin (in the presence factor, in addition to the ubiquitin-activating enzyme. Since of ATP and ubiquitin) was examined. There was a complete the ATP requirement of binding suggested the formation of a loss of proteolytic activity in the unadsorbed fraction when thiol ester intermediate between an enzyme and columnthe extract was applied in the presence of ATP. In the absence bound ubiquitin (17), we attempted to elute this factor with a of ATP, there was also a considerable decrease, although some high concentration of a thiol compound. Reticulocyte Fraction residual activity remained. In different experiments, this re- I1 was applied to the affinity column in the presence of ATP, sidual activity varied between 10 and 50% of the activity of and bound material was serially eluted with high salt, AMP untreated Fraction I1 (maximal variation). Since the ubiqui- + PP, (to elute ubiquitin-activating enzyme (17)), a buffer containing a high concentration of dithiothreitol, and a wash tin-activating enzyme is bound to the affinity column only in the presence of ATP (17), these results indicated that some at ph 9 (see Materials and Methods ). As shown in Table I, other factor(s) of the proteolytic system are also removed Experiment 1, the DTT eluate of the affinity column restored from Fraction I1 by the ubiquitin column, presumably by a the activity of the proteolytic system in the presence of the noncovalent interaction. KC1 eluate and the unadsorbed fraction. In this reconstituted In an attempt to recover the factor(s) bound to the affinity column in the absence of ATP, the column was sequentially 30 A- B. eluted with high salt and then by raising the ph to 9.0. As shown in Fig. 24, the high salt eluate of the affinity column restored the activity of the proteolytic system, when added to W Q, Untreated the unadsorbed fraction. Another part of column-bound activity, which was not displaced by high salt, could be eluted by the subsequent wash at ph 9. Control experiments indicated that neither the high salt eluate nor the ph 9 eluate had significant proteolytic activity by themselves, without the unadsorbed fraction (data not shown). A further control showed that no activity was bound to a Sepharose column which had no ubiquitin attached. Although the high salt eluate of the affinity column restored protein breakdown in the unadsorbed material of Fraction I1 applied to the column in the absence of ATP, it was not sufficient to do so with the unadsorbed fraction of chromatography performed in the presence of ATP (Fig. 2B). This indicated that in the presence of ATP, some further factors D Applied-ATP a KC1 eluate Applied-ATP Applied+ATP Fraction added (pg of protein) FIG. I. Binding of components of the proteolytic system to ubiquitin-sepharose in the presence or absence of ATP. 1-ml portions of Fraction I1 from reticulocytes were applied to 1-ml columns (0.5 X 5 em) of ubiquitin-sepharose in the presence of ATP (A), as described under Materials and Methods, or under similar conditions but with ATP omitted (A). Samples of the unadsorbed fractions or of untreated Fraction I1 (0) were assayed for the degradation of?%albuminin the presence of ubiquitinandatp, as described under Materials and Methods. Applied+ATP Breakthrough fraction (pg) Column eluate ( pg) FIG. 2. Elution of factorb) bound to the affinity column in the absence of ATP. A, reconstitution of activity by eluates of high salt or ph 9. Fraction I1 was applied to ubiquitin-sepharose in the absence of ATP as described in the legend to Fig. 1 and the column was washed with 3 column volumes of Buffer A (see Materials and Methods ) lackingatp.thecolumn was eluted with 6 column volumes of 1 M KC1 containing 50 mm Tris-HC1 buffer (ph 7.2), followed by 3 column volumes of 50 mm Tris-HC1 (ph 9.0) containing 2 mm dithiothreitol. The degradation of 251-albumin was determined in the presence of the unadsorbed fraction of the same column (120 pg of protein) and the indicated amounts of the KC1 eluate (0) or ph 9 eluate (0). B, lack of reconstitution by KC1 eluate with the unadsorbed fraction of extract chromatographed in the presence of ATP. Fraction I1 was applied to ubiquitin-sepharose columns in the presence or absence of ATP, as described in the legend to Fig. 1, and increasing amounts of the unadsorbed (Breakthrough) fractions were assayed in the presence of 23 pg of KC1 eluate.

4 miquitin-protein Ligase System 8209 system too, there was a complete requirement of protein breakdown for the presence of ubiquitin and ATP (data not shown). For convenience of reference, we shall term the three 30 factors eluted from the affinity column E, (ubiquitin-activating enzyme), E2 (factor eluted by DTT), and E3 (eluted with high salt or high ph). It seemed that some of the affinity column eluates contained mixtures of at least two of these factors. The lack of requirement for added El in the presence of the DTT eluate (Table I, Experiment 1) can be explained by the presence of considerable amounts of E, in this fraction, Elution Volume tml) which was not completely removed by the prior elution with E AMP and PPI (cf. Table 111). On the other hand, in the E lo presence of the ph 9 eluate, El stimulated protein breakdown without DTT eluate (Table I, Experiment l), suggesting that the ph 9 eluate contains E, as well as E3. To obtain a better separation between the three factors, eluates of the affinity column were subjected to gel filtration Fractim Mumbn chromato@aphy. When the DTT e uate was separated On a FIG. 3. Gel filtration chromatography of factor eluted with Cohmn of Sepharose 6B and column fractions were assayed dithiothreitol. 570 p1 of DTT eluate (containing 532 nanounits of for E, activity (protein breakdown in the presence of El, Ea, E, activity) were applied to a column (0.9 x 58 cm) of Sepharose 6B and the unadsorbed fraction), a single peak of an apparent (Pharmacia) equilibrated with 20 mm Tris-HC1 (ph 73, 1 mm molecular weight of around 35,000 was found (Fig. 3). By dithiothreitol, and 1 rng/ml of ovalbumin. Elution was with the above contrast, when the p~ 9 was separated on a similar buffer and fractions of 0.74 ml were collected at 4 C. Protein breakdown (0) was assayed in fraction samples of 5 p1 in the presence of column, two peaks of E2 activity were observed; in addition 180 pg of unadsorbed fraction, 33 pg of KC1 eluate, and 7.3 nanounits to the M r = 35,000 enzyme, there is a higher molecular weight of purified Conjugation of l251-~biquitin (0) was assayed as deform (MI E 250,000) of apparently similar activity (Fig. a). scribed under Materials and Methods, in samples of 2 pl in the Since the lower molecular weight form of E2 is well separated presence of 3.6 nanounits of El and 4.6 pg of iodoacetamide-treated from E, (M, = 210,000, Ref. 17) by gel filtration, this form ph 9 eluate. The ph 9 eluate was treated with iodoacetamide (5 mm) was mainly used for subsequent studies. With purified E2, at 25 c for 10 min, followed by the addition of excess (8 mm) dithiothreitol. Inset, estimation of molecular weight. Marker proteins: there was a nearly complete requirement of protein break- 1, alcohol dehydrogenase (M, = 150,000); 2, hemoglobin (M, = 64,000); down for El, when assayed in the Presence of KC1 eluate and 3, ovalbumin (M, = 43,000); 4, myoglobin (M, = 17,000). The arrow the unadsorbed fraction (Table I, Experiment 2). Assay of indicates the elution position of the enzyme. Sepharose 6B column fractions of the ph 9 eluate for E3 activity (protein breakdown in the presence of excess El, E,, the and unadsorbed material) showed a single peak with an TABLE I1 apparent molecular weight of approximately 3oo,000 (F~~. Conhwtion of ubiquitin with proteins requires the three affinitypurified factors of the proteoiytic system 4B)* The molecular size Of E3 derived from the Of Conjugation of 1251-UbiqUitin was determined as described under the affinity column, as determined by gel filtration, was Materials and Methods. Where indicated, enzymes were added at similar to that derived from the PH 9 eluate (data not shown). the following amounts: E,, 3.6 nanounits; E2 (pooled peak fractions Role of the Three Affinity-purified Factors in. the Conjuga- from Fig. 3), 10 pl; E3 (pooled peak fractions from Fig. 4B), 3 pl, In tion of Ubiquitin with Proteim-Trying to identify the func- Experiment 2, E3 was treated with iodoacetamide as described in the tions of E2 and E, we considered the possibility that they may legend to Fig. 3. be required for the ubiquitin-protein conjugation process. As Additions I-Ubiquitin conjugated pmol TABLE I Experiment 1 E, 0.7 Restoration of uctiuity of the proteolytic system by factors bound to E2 0.7 the affinity column in the presence of ATP E3 1.3 Fraction I1 was applied to ubiquitin-sepharose in the presence of ATP, and affinity chromatography was carried as out described under E2 + E3 4.3 Materials and Methods. The degradation of 1251-albumin was de- + E3 5.3 termined as described under Materials and Methods, in the pres- + E2 + E ence of 171 pgof the unadsorbed fraction. Where indicated, the Experiment 3, following amounts of affinity column eluates were added (micrograms iodoacetamide-treated 0.3 E37 of protein): KC1 eluate, 17.5; DTT eluate, 5.7; and ph 9 eluate, 5.2. iodoacetamide-treated 1.o Purified E, was supplemented at 14.6 nanounits, and purified E2 (cf. Ea, iodoacetamide-treated E2 1.2 Fig. 3) at 1.6 nanounits. Es, iodoacetamide-treated + E, + E2 7.1 Additions I-Albumin degraded % shown Table in 11, when El and E2 were incubated with Experiment 1 ubiquit,in and ATP, little if any formation of ubiquitin-protein KC1 eluate 5.4 conjugates could be detected. However, upon the supplemen- KC1 eluate El 4.7 KC1 eluate + tation of all three factors (purified by affinity chromatography DTT eluate 23.9 ph 9 eluate 4.7 and gel filtration), significant conjugation of ubiquitin with ph 9 eluate + E, 11.0 proteins was observed. partial The requirement for E, and E2 Experiment 2 in the presence of E3 is presumably due to contamination with KC1 eluate E, 4.2 E1 and incomplete separation of the high molecular weight KC1 eluate E, 5.8 form of E, from E3 on gel filtration (Fig. 4). However, E3 KC1 eluate + E, + E could be freed of residual E2 and El activities by treatment

5 Ubiquitin-Protein Ligase System A. Fa Cat ADH Hb Yb I 4 Y B. R 7 FIG.4. Gel filtration chromatography of factors eluted at ph 9. A, elution profile of E pl of ph 9 eluate (containing 933 nanounits of Ez activity) were applied to a Sepharose 6B column under conditions identical with those described in the legend to Fig. 3. The breakdown of 1251-albumin (0) was assayed in samples of 15 pl in the presence of 180 pg of unadsorbed fraction, 44 pg of KC1 eluate, and 7.3 nanounits of purified E,. Conjugation of 1251-ubiquitin (0) was assayed as described under Materials and Methods in fraction samples of 3 pl in the presence of 3.6 nanounits of E, and 2.3 pg of iodoacetamide-treated ph 9 eluate (see Fig. 3). B, elution profile of E pl of ph 9 eluate (containing 535 nanounits of E, activity) were separated on Sepharose 6B under conditions identical with those described above. Protein breakdown was assayed in samples of 20 pl, in the presence of 110 pg of unadsorbed fraction, 7.0 pg of DTT eluate, and 7.3 nanounits of E,. Conjugation of 1251-ubiquitin was determined in samples of 2 pl, in the presence of 3.6 nanounits of E, and 0.7 pg of DTT eluate. Markers (arrows): Fe, ferritin (M, = I I 4 6 lh 116 Minutes FIG. 5. Effect of iodoacetamide on the activity of the three enzymes. El (purified bygel filtration from AMP-PPi eluate, see Materials and Methods ), E2 (purified by gel filtration from DTT eluate, Fig. 31, and E3 (purified by gel filtration from ph 9 eluate, Fig. 48) were diluted in 20 mm Tris-HC1 (ph 7.2) containing 1 mg/ ml of ovalbumin and 0.1 mmof dithiothreitol to a concentration of 75 nanounits/ml. Following the addition of iodoacetamide (5 mm, final concentration), the samples were incubated at 37 C. At various time intervals, aliquots of 5 pl were transferred to the reaction mixture of the conjugation assay (see Materials and Methods ), which contained an %fold excess of dithiothreitol over iodoacetamide. The ubiquitin-conjugating activity of Ez was determined as described in the legend to Fig. 3, and that of E3 as described in the legend to Fig. 4B. The activity of E, was determined in the presence of0.67 nanounits of purified E2 and 2.3 yg of ph 9 eluate. activity of each of the three enzymes, provided that the other two are in excess. As shown in Fig. 3, the ubiquitin-conjugating activity of E2 derived from the DTT eluate of the affinity column coincided exactly with the proteolytic activity of this enzyme, assayed in fractions of the same Sepharose 6B column. A similar coincidence between the ubiquitin conjugation and protein breakdown activities of the two forms of E, derived from the ph 9 eluate (Fig. 4A) and of EB from the same eluate (Fig. 4B) were observed. Moreover, when E, was separated on a Sephacryl S-200 column, the peak of its activity assayed by ubiquitin-dependent PPI-ATP exchange reaction (17) coincided with the activity of this enzyme to stimulate protein breakdown (data not shown). These data indicate the identity of the three enzymes of the ubiquitin-protein ligase system with the corresponding factors participating in protein 480,000); Cat, catalase (M, = 240,000); ADH, alcohol dehydrogenase breakdown. (M, = 150,000); Hb, hemoglobin (M, = 64,000); Mb, myoglobin (M, = Purification of E2 and E3-The ubiquitin conjugation assay 17,000). (which is more accurate and sensitive than the proteolytic assay) was used to determine the extent of purification of E, with iodoacetamide. El is rapidly inactivated by iodoaceta- and EB by the affinity chromatography procedure. In Table mide (13), and so is E2 (Fig. 5). By contrast, E3 is much more 111, the distribution of Ez in various fractions of the affinity resistant to thi sulfhydryl blocking agent (Fig. 5). As shown column is compared with that of E,. It may be seen that most in Table 11, Experiment 2, following treatment of E3 with of eluted E, activity distributed about equally between the iodoacetamide, a virtually complete requirement for El and DTT eluate and the 9 ph eluate. However, the specific activity EP for ubiquitin conjugation was observed. Iodoacetamide- of E, in the DTT eluate was about 2-fold higher than that in treated E, (derived from the ph 9 eluate) also showed a the ph 9 eluate, and a more than 90-fold purification was complete requirement for E, and E2 in the stimulation of achieved in the former fraction. It should be mentioned that protein breakdown (data not shown). some EP activity eluted in other fractions well, as most notably To examine whether the threenzymes required for in the AMP-PP, eluate (containing bulk the of El). Therefore, ubiquitin-protein conjugation are indeed similar to those par- we used E, purified on a Sephacryl S-200 gel filtration column ticipating in protein breakdown, the gel filtration profiles of (which separates it from residual low molecular weight EP) the proteolytic and conjugating activities were compared. The throughout this study (see Materials and Methods ). ubiquitin conjugation assay can be used to determine the Table IV shows the purification of E3 and its distribution

6 Ubiquitin-Protein Ligase System 8211 TABLE 111 Distribution of E, and E2 in fractions of the affinity column 22 ml of Fraction I1 from reticulocytes were subjected to affinity chromatography as described under Materials and Methods. The activity of E2 was determined by the lz5i-ubiquitin conjugation assay (see Materials and Methods ) as described in the legend to Fig. 3, and the activity of E, as described in the legend to Fig. 5. Total activity Recovery Specific activity Purification Fraction Total protein El E2 E1 E2 EI Ez EI E2 mg nanounits % nanounits/mg (-fold) Fraction I ,060 3, KC1 eluate AMP-PPi eluate , , DTT eluate , , DH 9 eluate TABLE IV Distribution of E, in fractions of the affinity column 10 ml of Fraction I1 from reticulocytes were subjected to affinity chromatography as described under Materials and Methods. The activity of E, was determined by the?-ubiquitin conjugation assay (see Materials and Methods ), as described in the legend to Fig. 4B. Total pro- Total Recov- Specific Purifi- Fraction tein activity ery activity cation w units % u?z,& -fold Fraction I , Unadsorbed fraction 195 2, KC1 eluate AMP-PPi eluate DTT eluate ph 9 eluate TABLE V Binding of E, to ubiquitin-sepharose requires E, and ATP 0.5-ml portions of E2 (low molecular weight enzyme from the Sepharose 6B column of the ph 9 eluate (Fig. U)), containing 73.3 nanounits, were adjusted to 20 mm Tris-HC1 (ph 7.2), 1 mg/ml of ovalbumin, 0.2 mm dithiothreitol, and 5 mm MgCl,. Where indicated, 2 mm ATP or 350 nanounits of E, were added. The samples were applied to 1-ml columns (0.5 X 5 cm) of ubiquitin-sepharose equilibrated with buffers of identical composition to the corresponding samples. Affinity chromatography was carried out as described under Materials and Methods, except that all solutions contained ovalbumin (1 mg/ml), to minimize inactivation of the dilute enzyme. E2 activity was determined by the 1251-uhiquitin conjugation assay (see Materials and Methods ), as described in the legend to Fig. 3. Additions Ez activity recovered E~ ATP :;%:Fd KCI eluate DTT eluate p~ geluate % of applied in fractions of the affinity column. About one-half of Ea activity remained in the unadsorbed fraction in this prepara tion, and column-bound E3 eluted mainly in the KC1 eluate and ph 9 eluate fractions. However, the KC1 eluate contains a considerable amount of protein, apparently bound nonspe- TABLE VI cifically to the ubiquitin-sepharose column. Therefore, the Binding of E3 to ubiquitin-sephurose in the presence or absence of extent of purification of E3 in the high salt eluate is relatively A TP low, in contrast to a nearly 25-fold purification of E3 achieved 25O-pl portions of EB from the Sepharose 6B separation of the ph in the ph 9 eluate (Table IV). 9 eluate (Fig. 4B), containing 43.5 nanounits of activity, were applied Although considerable purification is achieved by the pres- to 1-ml ubiquitin-sepharose columns in the presence or absence of ent procedure, the preparations of E2 and E3 obtained are not ATP and E,, under conditions identical with those described in the homogenous. SDS-polyacrylamide gel electrophoresis of E2 legend Table V. Activity of E3 was assayed by the lzi-ubiquitin (DTT eluate further purified by gel filtration on Sephadex G- conjugation method, as described in the legend to Fig. 4B. 100) showed four Coomassie blue staining bands of M, = Ea activity recovered 28,000, 20,000, 16,000, and <10,000, while the preparation of Additions U ~ ~ ~ KCI : eluate ~ d zuz p~ 9 eluate E, (ph 9 eluate purified on Sepharose 6B) had three major protein bands (Mr = 200,000, 100,000, and 64,000) and nu- % of applied merous minor bands (not shown). The presence of these None impurities presumably accounts for the observation that even + ATP and 43.3 E, with the purified system, about one-half of ubiquitin conjugates formed are derived from endogenous protein substrates. in the presence of ubiquitin (data not shown). Therefore, we Both preparations were free of detectable nonspecific protease tested whether the re-binding of E2 (purified through the and ATPase activities. However, the preparation of E3 con- affinity and gel filtration columns) to the ubiquitin column tained some enzyme activity which degrades ubiquitin-protein requires E, as well as ATP. As shown in Table V, this indeed conjugates. The latter activity is inhibited by sulfhydryl re- was found to be the case. In the presence of El and ATP, agents, and thus E3 can be freed of the conjugate-degrading most E2 was bound to the column and recovered in the DTT enzyme by treatment with iodoacetamide. and ph 9 eluates, whereas upon the omission of either El or Requirements for the Binding of E2 and E3 to the Affinity ATP there was no significant binding of Ez, and most of the Column-We next asked in what form are E2 and E3 bound recovered activity was in the unadsorbed fraction. Similar to the affinity column. The general characteristics of the results were obtained with the high molecular weight form of binding of E2, i.e. the requirement for ATP, lack of displace- E2 (data not shown). These results suggest an E,-mediated ment of column-bound enzyme by high salt, and its elution binding of Ez to the ubiquitin column as a covalent intermeby high concentrations of DTT or increased ph, resemble diate (see Discussion ). those of El, which is bound to the affinity column as a covalent In contrast to E2, the binding of purified E, to the affinity thiol ester intermediate (17). However, E2 does not activate column did not require El or ATP (Table VI), suggestive of ubiquitin since purified E, had no ATP-PPI exchange activity noncovalent interactions. It is notable, however, that only a

7 8212 Ubiquitin-Protein Ligase System part of column-bound E3 can be eluted with high salt, while another part is eluted with high ph (Table IV), suggesting different types of interactions. To examine whether these represent two different species of E3, the fraction of enzyme eluted at ph 9 was applied again to ubiquitin-sepharose and its elution pattern was determined. As shown in Table VI, again a partition of E, between the high salt and ph 9 eluate was observed. Moreover, with E, derived from the high salt eluate, a similar partitioning between the above two fractions was found (data not shown). The results indicate, therefore, that this elution pattern is not due to distinct forms of the enzyme, but possibly represents different types of interactions of E3 with column-bound ubiquitin. It should be noted that ubiquitin is possibly bound heterogenously to the column both with regard to the site of attachment of lysine residues of ubiquitin and also relative to matrix structure. Transfer of Activated Ubiquitin from E, to E,-Attempting to clarify the roles of E2 and E3 in the ligation of ubiquitin with proteins, we considered the possibility that E2 may have a function in the transfer of activated ubiquitin to the site of amide bond formation. Such a possibility was initially suggested by the observation that the binding of E2 to ubiquitin- Sepharose requires El as well as ATP (Table V). Further evidence for the transfer of ubiquitin from E, to a thiol site on E2 was provided by the characteristics of the protection of these enzymes against inactivation by iodoacetamide. E, can be protected against iodoacetamide by a prior incubation with ATP and ubiquitin (Table VII, Experiment 1). This is presumably due to the formation of the thiol ester linkage be- TABLE VI1 Protection of E, and E2 against inactivation by iodoacetamide For Experiment 1 purified Et (see "Materials and Methods") was diluted to a concentration of 66.7 nanounits/ml in a solution of 50 mm Tris-HCI (ph 7.61, 1 mm dithiothreitol, 1 mg/ml of ovalbumin, 50 mmm&12, and 2 units/ml of inorganic pyrophosphatase, in a final volume of 50 pl. Where indicated, 2 mm ATP or 1 p~ unlabeled ubiquitin was supplemented. The mixtures were incubated at 25 "C for 5 min before the addition of iodoacetamide (4 mm) and incubation for a further 10 min. Samples of 2 pl were then transferred to the reaction mixture of the 12SI-ubiquitin conjugation assay (see "Materials and Methods"), which contained a 25-fold excess dithiothreitol over iodoacetamide. The reaction mixtures were adjusted to equal amounts of ubiquitin (54 pmol), so that the specific radioactivity of 125 I-ubiquitin was identical in all samples. Activity of E, was assayed in the presence of 0.67 nanounits of purified E2 (Fig. 3) and 4.6 pg of iodoacetamide-treated ph 9 eluate (Fig. 3). Results are expressed as the percentage of the activity of a control sample which was treated similarly, except that iodoacetamide was mixed with a IO-fold molar excess of dithiothreitol prior to its addition. For Experiment 2, a purified preparation of E2 (see Fig. 3) was diluted to a concentration of 56 nanounits/ml and incubated under conditions similar to those described for Experiment 1. Where indicated, 2 mm ATP, 1 ym ubiquitin, or 150 nanounits/ml of purified E1 were supplemented to the incubation prior to the addition of iodoacetamide. E2 activity was assayed in the presence of 3.6 nanounits of E, and 4.6 pg of iodoacetamide-treated ph 9 eluate. For Experiment 3, experimental conditions were similar to those of Experiment 2, except that the low molecular weight peak of E2 from the gel filtration column of the ph 9 eluate (see Fig. 4A) was used, at a concentration of 76.7 nanounits/ ml. Activity remained Additions E1 El tween ubiquitin and the thiol site of El. E, can also be % of control protected against iodoacetamide inactivation, but this re- Experiment 1 quires a preincubation with El, in addition to ubiquitin and El ATP 6 ATP (Table VII, Experiments 2 and 3). This suggests the 19 E, + ubiquitin transfer of ubiquitin, activated by E, in the presence of ATP, E,+ATP+Ub 82 to an iodoacetamide-sensitive thiol site of E2. Experiment 2 The thiol ester El-ubiquitin is sufficiently stable to be E2 E, ATP 3 E, separated by SDS-polyacrylamide gel electrophoresis when E, ubiquitin 0 E2 + E, + ATP + ubiquitin 96 electrophoresis is performed at 4 "C (17). We therefore Experiment 3 searched for a possible transfer of lz5i-ubiquitin from E1 to E2 on gels run under similar conditions. In the experiment shown El ATP ubiquitin E, + ATP + ubiquitin + E, 5 86 in Fig. 6, lanes 1-3, 1251-~biq~itin was first incubated with El formation of El-ubiquitin thiol ester (subunit size, M, = 105,000, Ref. 17). The specific radioactivity of residual free in the presence of ATP (Fig. 6, lane I), resulting in the of was more labile than that of ubiquitin-protein conjugates. When the sample was boiled in the presence of mercap- E2 ubiquitin was then lowered 1,000-fold by the addition of a toethanol prior to electrophoresis, ubiquitin was released from large excess of unlabeled ubiquitin, following which E2 was all bands (Fig. 6, lane 7). This is in contrast to the stability added to the incubation. As shown in Fig. 6, lane 2, there was a marked decrease in the amount of El-bound l2'1-ubiquitin, with the appearance of four new bands of lower molecular weight. When unlabeled ubiquitin was added before El and E2, none of the bands was significantly labeled (Fig. 6, lane 3), indicating that the isotopic dilution of lz5i-ubiquitin was of the amide linkage of ubiquitin-protein conjugates in such treatment (7). In addition, ubiquitin was also released from its linkage to E2 by treatment with 0.1 N NaOH (but not with 1 N formic acid) or by treatments with 1 M hydroxylamine at ph 8 or 1% mercuric acetate (data not shown), under conditions identical with those used to characterize the E1-ubiquitin adequate, and thus the new bands of Ez originate from El- thiol ester linkage (12). These data indicate that activated bound "'I-ubiquitin. Similar transfer of activated ubiquitin ubiquitin is transferred from El-ubiquitin to thiol ester interfrom E,-ubiquitin to the four bands of E2 was observed when mediates of ubiquitin with E,. In experiments of similar ATP was removed with hexokinase and glucose prior to the design, no transfer of activated ubiquitin from E1 to E3 addition of E2 (data not shown). The apparent molecular (without E2) was observed. weight of the four bands of E,, designated bands 1-4 in Transfer of Activated Ubiquitin to Conjugate Formation in increasing molecular size, are 21,500, 23,000, 32,000, and the Presence of E3-We next asked whether the EP-ubiquitin 34,000, respectively. In addition, a region of diffuse radioac- thiol esters can be donors for conjugate formation. In the tivity between band 1 and free ubiquitin can be seen, which experiment shown in Fig. 6, lanes 4-6, E, was first incubated may represent ubiquitin released from enzyme-bound forms with '9-ubiquitin, ATP, and a smaller amount of El. AS during electrophoresis. All four bands of EP were present in shown in Fig. 6, lane 4, the expected thiol esters of EI and EP different preparations of E2, although in some preparations were formed. ATP was then removed with hexokinase and bands 1 and 2 were much more prominent than bands 3 and glucose, and E3 was added for a further 10-min incubation (in 4. the presence of oxidized RNase as the conjugation substrate). The linkage of ubiquitin to the low molecular weight bands As seen in Fig. 6, lane 5, there was a marked loss of lz5i-

8 Ubiquitin-Protein Ligase System 78 Cont. P \ 8213 quantitation, the various lanes were cut into4-mm piecesand radioactivity in the different bands was estimated by y counting. A small amount of high molecular weight contaminants, present in this preparation of "'1-ubiquitin, was subtracted from the corresponding positions. In this experiment, the totalamount of 1251-ubiquitin-proteinconjugates was0.40 pmol, as compared to 0.06 pmol of ubiquitin lost from E,ubiquitin, and 0.36 pmol lost from all fourforms of EZubiquitin. This indicates thatthe different forms of E2ubiquitin were the main donors for conjugate formation in the presence of E3. DISCUSSION ubiquitin from bands 1 and 2 and a partial decrease in bands 3-4, concomitant with the appearance of numerous high molecular weight bands. These new bands are ubiquitin-protein conjugates, as shown by the finding that they are resistantto boiling in the presence of SDS and mercaptoethanol (Fig. 6, lane 8).A control (Fig. 6, lane 6 ) showed that theaddition of a similar amount of hexokinase and glucose prior to the addition of all three enzymes prevented the formation of conjugates, indicating that ATP was sufficiently removed under these conditions in the transfer experiment. It should be noted that activated ubiquitin bound to E,, as well as to the different forms of E2-ubiquitin, were lost during Protein- Ub the formation of ubiquitin-protein conjugates (Fig. 6, compare Ub*ATP 2- S-Ub Conjugate lanes 4 and 5). The question arose which enzyme-ubiquitin FIG.7. Proposed sequence of events inthe ubiquitin-protein thiol ester is the main source for conjugate formation. For ligase system. See the text. Ub,ubiquitin. AMp*wixE;i;+- "") :c ~ FIG.6. Transfer of activated ubiquitin from Elto Ezand to conjugate formation in thepresence of Es.All incubations contained, in a final volume of 20 pl, 50 mm Tris-HCI (ph 7.2), 5 mm MgCI2, 0.1 mm ATP, 0.2 mm dithiothreitol, 0.1 unit of inorganic pyrophosphatase, 10 pgof oxidized RNase, and 1.84 pmol of '%Iubiquitin (9900cpm/pmol). Lanes 1-3, transfer of activated ubiquitin from El to E2.Lune 1, incubated with E, (0.46 nanounits) a t 37 "C for 5 min. Lane 2, incubated with El as in lane 1, then 2000 pmol of unlabeled ubiquitin were added, followed by the addition of purified E2 (0.6 nanounits), and a further incubation of 5 min. Lane 3, 2000 pmol of unlabeled ubiquitin were added before the addition of the above amounts of El and E2,and the mixture was incubated for 10 min. Lanes 4-8, transfer of E,-bound ubiquitin to conjugate formation in the presence of En. Lane 4, incubated a t 37 "C for 5 min in the presence of E, (0.073 nanounits) and E2 (0.6 nanounits); lane 5, incubated as in lane 4, then hexokinase (1 unit) and 2-deoxyglucose (10 mm) were added for a further 3-min incubation. This was followed by the addition of 0.46 nanounits of En(from the Sepharose 6B peak of the ph 9 eluate, treated with 5 mm iodoacetamide for 15 min a t 37 "C) and incubation was continued for a further 5 min. Lane 6, hexokinase and deoxyglucose, at theabove amounts, were incubated with the reaction mixture for 3 min before the supplementation of E,, E2, and E3(at theabove amounts) and a further incubation of 10 min. Samples 1-6 were treated with 0.5% SDS a t 0 "C for 30 min before electrophoresis. Lanes 7 and 8, incubations identical with lanes 4 and 5,respectively, but thesamples were boiled for 3 min, with 2% SDS and 3% mercaptoethanol prior to electrophoresis. SDS-polyacrylamide gel electrophoresis was performed as described previously (17)on 12.5% polyacrylamide running gel and 6%stacking gel a t 30 ma for 3.5 h a t 4 "C. The gel was stained, destained, dried, and radioautographed as described (7). EI-Ub,El-ubiquitin thiol ester; Cont., contaminations in the preparation of '&I-ubiquitin; 1-4, different E2-bound forms of 9-ubiquitin. The present study was initiated by an examination of the components of the ubiquitin-atp system which can be isolated by affinity chromatography on ubiquitin-sepharose. Since several enzymes in this pathway may have specificsites for ubiquitin, it was expected that further components, in addition to the ubiquitin-activating enzyme (17), may bind to the affinity column. In fact, two further factors of the proteolytic system were isolated by this method: E2,which binds to the affinity column in the presence of ATP, and ES, the binding of which does not require ATP. We then found that E2and En,in concert with E,, are participating in the conjugation of ubiquitin with proteins (Table 11).We propose to designate the three enzymes as components of the ubiquitinprotein ligase system. The identity of the components of the ligase system with the factors of the proteolytic system was indicated by the coincidence of the corresponding activities across gel filtration columns (Figs. 3 and 4). These results provide further support for the role of the conjugation of ubiquitin in protein breakdown. Since the purified ubiquitin-activating enzyme does not carry out conjugation by itself (18),the existence of a further enzyme (which would catalyze amide bond formation between ubiquitin and proteins) was expected, but the observed requirement for twodistinct additional enzymes wassurprising. The present data indicate that the role of E2 might be the transfer of activated ubiquitin to the site of amide bond formation, and the proposed sequence of events is depicted in Fig. 7. According to this scheme, activated ubiquitin bound via its COOH terminus to the thiol site of E, is first transferred to another sulfhydryl site one2.the first clue for such a transfer process was the observation that thebinding of E2 to the ubiquitin column requires ATP, as well as E, (Table V). This canbe explained by the assumption that E2replaces column-bound El in a thiol ester linkage, although the possibility that E2 is bound through E, could not be ruled out. A more direct proof for the involvement of thiol groups on Ez in this process is the finding that protection of E2 against inactivation by iodoacetamide requires preincubation with E,, in the presence of ubiquitin and ATP (Table VII). Finally, transfer of activated "'I-ubiquitin from E, to the different forms of E2can be directly demonstrated by polyacrylamide gel electrophoresis (Fig. 6), and thelinkage of ubiquitin to all forms of E2 has the stability characteristics of a thiol ester bond. It should be noted that thiol transesterification of

9 tin-protein 8214 activated carboxyl residues occurs in other biosynthetic processes such as in fatty acid synthesis (21) or in the more closely analogous synthesis of peptide antibiotics (22). In both of these cases, reversible transfer of thiol esters is between enzyme-bound cysteine and 4'-phosphopanthetheine residues. It may well be that a pantetheine group has an analogous role in the ubiquitin-ligase system. We find that ubiquitin is further transferred from E,-bound thiol esters to stable conjugates in the presence of E3 (Fig. 6). The function of EB may thus be the catalysis of amide bond formation between ubiquitin and proteins. It is possible, however, that E3 has an essentially required structural role, in which case the ligase function may reside in one of the earlier components. Another unsolved problem is the significance of the multiple forms of E,. The high molecular weight form of E, does not seem to be an E2. E3 complex, since its molecular weight is slightly lower than that of E3 (Fig. 4). It might be a multimer of low molecular weight E,, or an isoenzyme that carries out a similar function. In addition, low molecular weight E, is further composed of several different proteins which bind ubiquitin (Fig. 6). The different E,-ubiquitin bands do not seem to be incompletely dissociated subunits of a single enzyme, since their treatment with increasing concentrations of SDS or incubation with 0.5% SDS at 37 "C for prolonged time periods did not convert the higher molecular weight bands to the lower bands, but rather a uniform loss of '251-ubiquitin from all bands of E, was observed (data not shown). In addition, the apparent molecular weight of the different E,-ubiquitin bands does not fit the assumption that they consist of increasing numbers of ubiquitin residues bound to a single enzyme subunit. It is possible that some of the bands are nonspecific thiol esters of ubiquitin with proteins that contaminate the preparations of E,. This may be the case with bands 3 and 4, which are present in variable amounts and are only partially transferred to conjugates in the presence of E3 (Fig. 6). It is also possible, however, that the different subspecies of E, represent a family of enzymes of related function, but of different specificities. For example, the attachment of successive molecules of ubiquitin to the protein substrate may occur at different sites of the ligase, each specific for a particular type of lysine residue; the different subspecies of E, may then transfer ubiquitin to the corresponding specific ligation sites. These and other questions, Ligase System such as the specificity and control of the ubiquitin-ligase system, remain for future investigation. Acknowledgments-A part of this work was done during the stay of A. H. at the Institute for Cancer Research, Philadelphia. We thank Dr. Irwin A. Rose for heipful suggestions. The expert technical assistance of Clara Segal is gratefully acknowledged. REFERENCES 1. Simpson, M. V. (1953) J. Bwl. Chem. 201, Hershko, A., and Tomkins, G. M. (1971) J. Biol. Chem. 246, Hershko, A., Ciechanover, A., and Rose, I. A. (1979) Proc. Nutl. Acad. Sci. U. S. A. 76, Ciechanover, A., Hod, Y., and Hershko, A. (1978) Biochem. Biophys. Res. Commun. 81, Ciechanover, A., Elias, S., Heller, H., Ferber, S., and Hershko, A. (1980) J. Bwl. Chem. 255, Wilkinson, K. D., Urban, M. K., and Haas, A. L. (1980) J. Bwl. Chem. 255, Ciechanover, A., Heller, H., Elias, S. Haas, A. L., and Hershko, A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, Hershko, A., Ciechanover, A., Heller, H., Haas, A. L., and Rose, I. A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, Hershko, A,, and Ciechanover, A. (1982) Annu. Rev. Biochern. ~ ~~ 51, Hershko, A,, Eytan, E., Ciechanover, A., and Haas, A. L. (1982) J. Biol. Chem. 257, Chin, D. T., Kuehl, L., and Rechsteiner, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, Ciechanover, A., Heller, H., Katz-Etzion, R., and Hershko, A. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, Haas, A. L., Warms, J. V. B., Hershko, A., and Rose, I. A. (1982) J. Biol. Chem. 257, Haas, A. L., and Rose, I. A. (1982) J. Biol. Chem. 257, Hershko, A., Ciechanover, A., and Rose, I. A. (1981) J. Biol. Chem. 256, Busch, H., and Goldknopf, I. A. (1981) Mol. Cell. Biochem. 40, Ciechanover, A., Elias, S., Heller, H., and Hershko, A. (1982) J. Biol. Chem. 257, Hirs, C. H. W. (1955) J. Biol. Chem. 219, Hershko, A., Heller, H., Ganoth, D., and Ciechanover, A. (1978) in Protein Turnover and Lysosome Function (Segal, H. L., and Doyle, D. J., eds) pp , Academic Press, New York 20. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, Lynen, F. (1972) Biochem. SOC. Symp. 35, Lipmann, F. (1971) Science (Wash. D. C.) 173,