the protein substrate binding site of the ubiquitin-protein ligase

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Vol. 261, No. 26, Issue of September 15, pp. 11992-11999 1986 Printed in irS.A.

The Protein Substrate Binding Site of the Ubiquitin-Protein Ligase System*

(Received for publication, February 3, 1986)

Avram Hershkoz, Hannah HellerS, Esther Eytan, and Yuval ReissS From the Unit of Biochemistry, Faculty of Medicine, Technion-Zsrael Institute of Technology, Haifa, Israel and the Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 191 11

In order to gain insight into the mechanisms that determine the selectivity of the ubiquitin proteolytic pathway, the protein substrate binding site of the ubiquitin-protein ligase system was identified and examined. Previous studies had shown that the ligase system consists of three components: a ubiquitin-activating enzyme (E,) , ubiquitin-carrier protein (EZ), and a third enzyme, E3, the mode of action of which has not been defined. E3 from rabbit reticulocytes was further purified by a combination of affinity chromatography, hydrophobic chromatography, and gel filtration procedures. A 180-kDa protein was identified as the subunit of E3. Two independent methods indicate that E3 has the protein binding site of the ubiquitin ligase system. These are the chemical cross-linking of IZSI- labeled proteins to the E3 subunit and the functional conversion of enzyme-bound labeled proteins to ubiquitin conjugates in pulse-chase experiments. The trapping of E3-bound protein for labeled product formation was allowed by the slow dissociation of E3*protein complex.

The specificity of binding of different proteins to E3, examined by both methods, showed a direct correlation with their susceptibility to degradation by the ubiquitin system. Proteins with free a-NH2 groups, which are good substrates, bind better to E3 than corresponding proteins with blocked NH, termini, which are not substrates. Oxidation of methionine residues to sulfoxide derivatives greatly increases the susceptibility of some proteins to ligation with ubiquitin, with a corresponding increase in their binding to E3. However, a protein derivative which was subjected to both amino group modification and oxidation binds strongly to the enzyme, even though it cannot be ligated to ubiquitin. It thus seems that the substrate binding site of E3 partic- ipates in determining the specificity of proteins that enter the ubiquitin pathway of protein degradation.

Intracellular protein breakdown is a highly selective process in which specific proteins are degraded at widely divergent rates. At least part of this selective degradation is carried out by the ubiquitin (Ub’) proteolytic pathway. In this pathway,

* Supported by United States Public Health Service Grant AM- 25614 and a grant from the United States-Israel Binational Science Foundation. 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 American Cancer Society Grant BC-414 to Dr. Irwin A. Rose.

The abbreviations used are: Ub, ubiquitin; Me-, reductively methylated; Ox-, oxidized with performic acid; MetO-, methionine residues oxidized to sulfoxides; DTT, dithiothreitol; SDS, sodium dodecyl sulfate.

protein substrates are first ligated to the polypeptide Ub and then degraded by a system which specifically attacks Ub- conjugated proteins (for reviews, see Refs. 1-3).

The question arises of how specific proteins are selected by the Ub ligation system to be committed to degradation. The answer must lie both in specific features of protein structure which are recognized and in the mode of action of the Ub ligase system which enables it to recognize such protein structures. In a previous study we found that a free NH2- terminal a-NHz group of the protein substrate is an important structural determinant for its degradation by the Ub system (4). In the present investigation, we concentrated on the problem of which component of the Ub-protein ligase system contains the binding site for the protein substrate and the properties of this site. It appeared reasonable to assume that at least some initial selection of protein structures suitable for Ub ligation would occur at such a binding site.

The Ub-protein ligase system consists of three enzyme components: a Ub-activating enzyme (El), which has been thoroughly investigated (5-7); Ub-carrier proteins (E2), which accept activated Ub from El (8, 9); and a third enzyme, E3, the mode of action of which was not defined except for the observation that it is required for the final transfer of Ub from E2 to amide bond formation with proteins (8). Other types of Ub-protein ligase systems have been described, such as the E3-independent transfer of Ub from certain species of Ez to some basic proteins (9). Still another Ub conjugation system exists in the case of certain proteins that require tRNA for degradation and Ub ligation (10, 11). The mode of action of tRNA has not yet been elucidated.

With regard to the question of the protein binding site of the major Ub ligase system involved in protein breakdown, the possibilities were that either E2, or E3, or both, are involved in substrate protein binding. In this study we show that E3 contains the protein binding site. Some properties of the binding process and its specificity have been examined.

EXPERIMENTAL PROCEDURES

Materials-Cytochrome c from horse heart (Type VI) and from Saccharomyces cereuisiae (Type VIII), enolase from bakers’ yeast (Type 111), chicken egg white lysozyme (Grade I, 3x crystallized), bovine pancreatic RNase A (Type XI1 A), Ox-RNase, and chicken egg ovalbumin (Grade V) were obtained from Sigma. Crystalline rabbit muscle enolase was from Boehringer Mannheim. Ubiquitin was purified from human erythrocytes as described (12, 13). All of these proteins were found to be essentially homogeneous by SDS- polyacrylamide gel electrophoresis.

Phenyl-Sepharose CL-4B was obtained from Sigma, Ultrogel AcA 34 from LKB Instruments, and hydroxylapatite (Bio-Gel HT) from Bio-Rad. Bis(sulfosuccinimidy1)suberate was from Pierce Chemical Co. and hematoporphyrin from Sigma. Molecular mass standards for SDS-polyacrylamide gel electrophoresis (Sigma) were (kDa) myosin, 205; @-galactosidase, 116; phosphorylase b, 97; bovine serum albumin, 66; ovalbumin, 45.

Protein Modifications-Reductive methylation of Ub and lysozyme was carried out as described previously (4,14). Modification of amino

11992

Protein Binding Site of Ubiquitin Ligase 11993

groups was >95% in all cases, as determined with fluorescamine (15). Specific oxidation of methionine residues to methionine sulfoxide

was carried out by dye-sensitized photooxidation in acid, essentially as described by Jori et al. (16). Briefly, proteins at a concentration of 2 mM in 70% (v/v) acetic acid were mixed with an equal volume of 2 mM hematoporphyrin in the same solvent. The solution was exposed to illumination by four 100-watt light bulbs placed on four sides of a transparent water bath at a distance of 30 cm. A gentle stream of oxygen was passed through the solution, and the reaction was carried out for 60 min at 25 "C. Hematoporphyrin was removed by a column of Sephadex G-25 equilibrated with 0.2 N acetic acid. The preparation was dialyzed overnight against water and lyophilized. Oxidation of all methionine residues was essentially complete, as found by the resistance of oxidized proteins to cleavage by cyanogen bromide.

Enzyme Purification-E,, Ezr and E3 were purified from extracts of rabbit reticulocytes by a slight modification of the previously described affinity chromatography procedure (8). Fraction I1 was first applied to Ub-Sepharose (approximately a 20 mg of Ub/ml of swollen gel) in the absence of ATP at a ratio of fraction I1 to a column of 1:l (by volume). By this method, Ea free of Ez was obtained in the pH 9 eluate (8). The unadsorbed fraction, which was diluted 1.5-fold relative to fraction 11, was applied again to Ub-Sepharose in the presence of ATP at a ratio of 3:l (by volume), and E,, Ez, and residual E3 were isolated by the sequential elution protocol described earlier (8). In both cases, the pH 9 eluate was brought to 2-3% of the starting volume of fraction 11. El and E2 (low-molecular weight form eluted with DTT) (8) were further purified by gel filtration chromatography as described (8) except that a column (1 X 50 cm) of Ultrogel AcA 34 was used. Activities of all three enzymes were determined by the rapid quantitative assay for "'I-Ub conjugation described earlier (8). A unit of activity is defined as the amount of enzyme that converts 1 pmol of Ub to conjugates per min.

Ea was further purified by hydrophobic chromatography as follows. A 1-ml column (0.5 X 5 cm) of phenyl-Sepharose was equilibrated with 250 mM potassium phosphate (pH 6.7) containing 1 mM DTT. 1 ml of pH 9 eluate (containing approximately 1.5-2 mg of protein and 330-500 microunits of E3 activity) was mixed with 10 ml of the above buffer and applied to the column. The unadsorbed fraction (fraction A) was collected. The column was successively eluted with 10-ml portions of the following solutions, all of which contained 1 mM DTT: 10 mM potassium phosphate, pH 6.7 (fraction B); 5 mM potassium phosphate, pH 6.7 (fraction C); 1 mM Tris-HC1, pH 7.6 (fraction D); and 1 mM Tris-HC1, pH 7.6, containing 50% ethylene glycol (fraction E). All fractions were collected into tubes containing 1 mg of ovalbumin. The fractions were concentrated by centrifuge ultrafiltration with CF-25 Centriflo membrane cones (Amicon Corp.), and buffers were changed by two successive lo-fold dilutions with 20 mM potassium phosphate, pH 7.4, containing 1 mM DTT, followed by ultrafiltration in the same cone. The final volume of all fractions was approximately 0.3 ml.

Cross-linking Conditions-Reaction mixtures contained, in a volume of 20 pl, 25 mM potassium phosphate, pH 7.4, 3.3 microunits of E3 (purified through the phenyl-Sepharose step), approximately 1 pg of lZ5I-labeled protein (1.5-3 X 105 cpm), and 0.5 mg/ml ovalbumin. The addition of ovalbumin was necessary to prevent nonspecific adsorption of 'Z51-labeled proteins. Ovalbumin is not a substrate for Ub ligation (8), and at concentrations up to 2 mg/ml, it did not interfere with the cross-linking of '=I-labeled proteins to Ea. The mixtures were allowed to stay at 0 "C for 30 min, following which 2 pl of 0.5 mM bis(sulfosuccinimidy1)suberate (dissolved in ice-cold phosphate buffer immediately prior to use) was added. After an additional 15 min at 0 "C, the reaction was stopped by the addition of 2 pl of 1 M ethanolamine-HCl, pH 9.0. The samples were mixed with electrophoresis sample buffer (containing 2% SDS and 3% 2- mercaptoethanol), boiled for 5 min, and resolved by electrophoresis on an 8% SDS-polacrylamide gel (17). The gels were stained, dried, and autoradiographed.

"Pulse-Chase" Experiments-Unless otherwise stated, the ''pulse" mixture contained, in a volume of 10 pl, 40 mM Tris-HCl, pH 7.6, 2 mM DTT, 1 mg/ml ovalbumin, 2.0 microunits of E3, and 0.15 pg of '251-lysozyme (approximately 7 X 10' cpm). E3 was treated prior to use with 5 mM iodoacetamide at 25 "C for 10 min to inactivate isopeptidases (see Ref. 8). Following preincubation at 37 "C for 5 min, the pulse mixture was rapidly mixed with 10 p1 of "chase" mixture containing 40 mM Tris-HC1, pH 7.6, 2 mM DTT, 2 mM ATP, 10 mM MgClZ, 5 pg Me-Ub, 0.7 microunits of E,, 0.5 microunits of E2, and 60 pg of unlabeled lysozyme. Unlabeled lysozyme used for chase experiments was subjected to iodination with unlabeled NaI and chloramine T, under conditions identical to those used for radioio&-

nation. The chase mixture was also preincubated at 37 "C for 5 min, prior to mixing. Rapid mixing was obtained by holding the reaction tube on a Vortex mixer while injecting the chase mixture; by this method, mixing was complete within 1 s. Following further incubation at 37 "C for the time periods indicated in the figure legends, the reaction was stopped by the addition of 20 pl electrophoresis sample buffer (containing 2% SDS final concentration). The samples were electrophoresed on a 12.5% SDS-polyacrylamide gel.

RESULTS

Purification of E3-We have previously purified E3 partially by affinity chromatography of reticulocyte extract on Ub- Sepharose, followed by elution at pH 9 (8). This procedure is not specific for Ea, and other Ub-binding proteins are also eluted at pH 9. These include a Ub-COOH-terminal hydrolase (18, 19) and at least three different isopeptidases? As shown in Fig. lA, lane I, the pH 9 eluate of the affinity column contains numerous proteins. One way to identify which of these is a subunit of E3 is to examine the coincidence of protein bands with E3 activity upon further purification. We noted previously that E3 has a relatively low affinity for Ub, and under conditions optimized for affinity purification about one-half of E3 activity remains in the unadsorbed fraction (8). When the unadsorbed fraction is applied again to Ub-Sepha- rose, a significant part of E3 activity is bound and can be eluted with pH 9 buffer. Analysis of this preparation by silver staining (Fig. lA, lane 2) shows that at least six protein bands are reduced or absent, as compared to the first pH 9 eluate. These are presumably proteins which have higher affinity than E3 for Ub-Sepharose and are thus more completely removed by the first passage on the affinity column. Since the samples analyzed contained equal amounts of E3 activity, the E3 subunit has to be one of the other proteins, equally present in both preparations.

E3 from the pH 9 affinity eluate was further purified by hydrophobic chromatography on phenyl-Sepharose. The enzyme was strongly bound to the hydrophobic column and was eluted with a marked reduction of ionic strength (Fig. 1B). Analysis of the protein composition of various column fractions (Fig. lA, lanes A-E) showed that several proteins were eluted at higher ionic strength (fraction B), including a major protein of 100 kDa. Some of these may be isopeptidases, since about 60% of isopeptidase activity is eluted in this fraction (data not shown). On the other hand, another major protein of 180 kDa coincided with E3 activity, eluting mainly in fraction D.

The possibility that the 180 kDa protein is a subunit of E3 was examined by coincidence in chromatography on Ultrogel AcA 34. On gel filtration columns, E, elutes at M, - 300,000 (8). As shown in Fig. 2, the 180-kDa major band co-eluted with E3 activity on the gel filtration column (peak center, fractions 27-29). Several minor protein bands were not in coincidence with E3 activity (Fig. 2B). Similar coincidence of the 180-kDa protein with Ea activity was observed in hydroxylapatite chromatography, where the enzyme eluted with 100- 150 mM phosphate concentrations (data not shown). In none of these procedures was a homogeneous preparation of E3 obtained, but the 180-kDa protein was predominant over other reticulocyte proteins following the gel filtration step (Fig. 2B). Carrier ovalbumin could be removed by a second chromatography on phenyl-Sepharose, which does not adsorb ovalbumin (not shown). These findings suggest that the 180- kDa protein is the subunit of E3.

Cross-Linkingof Protein Substrates to E3 Subunit-We next examined the possible binding of substrates to purified E3 by a cross-linkage method. The enzyme was preincubated with various lZ5I-labeled proteins and then subjected to the action

E. Leshinsky and A. Hershko, unpublished observations.

11994 Protein Binding Site of Ubiquitin Ligase

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Fraction FIG. 1. Purification of Es by hydrophobic chromatography.

A , silver staining (30) of E, preparations separated on 8% SDS- polyacrylamide gel. Lane I, 0.33 microunits (1.7 pg of protein) of the first pH 9 eluate of Ub-Sepharose; lane 2, 0.37 microunits (1.4 pg of protein) of the second pH 9 eluate (see "Materials and Methods"); lanes A-E, 1 pl of corresponding fractions of hydrophobic chromatography of the second pH 9 eluate (see "Materials and Methods" for details of hydrophobic chromatography and designation of fractions). Numbers (kDa) on the left side indicate the position of molecular mass markers. Arrows next to lane 2 indicate the position of protein hands that are decreased or absent in the second pH 9 eluate. Arrowhead on the right side indicates the position of the 180-kDa protein. 0 0 , ovalbumin carrier. R, E, enzymatic activity in fractions of hydrophobic chromatography. Results are expressed as the percentage of E, activity applied, which was 420 microunits.

of the water-soluble bifunctional agent bis(su1fosuccin- imidy1)suberate (20, 21). As shown in Fig. 3, bis(su1fo- succinimidy1)suberate caused considerable aggregation and nonspecific cross-linkage of '2sI-labeled proteins in the absence of Es. However, in the presence of Ea, a prominent high- molecular weight cross-linkage product of labeled protein substrates was formed. The molecular mass of the E3-specific product (approximately 190-200 kDa) fits the assumption that it consists of a single molecule of '2sI-labeled protein linked to the 180-kDa E3 subunit. Examination of the cross- linkage of different labeled proteins to the Ea subunit revealed

I n B, 23 2 S 2 7 2 8 7 9 31 33 -0rtgm

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Fraction Number FIG. 2. Chromatography of E3 on Ultrogel AcA 34. 200 pl

(23.3 microunits of E, activity) of fraction D of the hydrophobic column (see Fig. 1) was applied to a column (0.7 X 30 cm) of Ultrogel AcA 34 equilibrated with 20 mM Tris-HCI, pH 7.2, 1 mM DTT, and 1 mg/ml ovalbumin. Fractions of 0.2 ml were collected. A , E, activity was determined in samples of 5 pI of column fractions. R, silver staining of samples of 20 pI of column fractions separated on an 8% SDS-polyacrylamide gel. Fraction numbers are indicated a t the top. On the left side are shown the positions of molecular mass markers (kDa). Arrowhead, position of the 180-kDa protein. Ou, ovalhumin carrier.

a general correlation to their susceptibility to the action of the Ub system. Thus, yeast cytochrome c and lysozyme, which have free NH2 termini and are good substrates, are efficiently cross-linked to E:+ On the other hand, equine cytochrome c (which has a blocked a-NH2 group and is not a substrate for Ub conjugation and protein breakdown (4)) is cross-linked to a much lesser extent. Modifications of RNase A which increase its susceptibility to conjugation with Ub (see below) also increase the extent at which it is found to be cross-linked to the E3 subunit (Fig. 3).

These results may indicate that proteins that are good substrates bind tightly to a specific site of E,, whereas poor substrates bind less tightly, or perhaps in a manner that does not allow cross-linking to occur under these conditions. We therefore examined the effect of unlabeled proteins on the cross-linkage of "'I-labeled yeast cytochrome c to &. As seen in Fig. 4, unlabeled yeast cytochrome c prevented the cross- linkage of the labeled substrate, whereas a similar excess of equine cytochrome c had much less of an effect. Derivatives of RNase A that are good substrates (see below) also compete well on the cross-linkage of labeled substrate (Fig. 4). The only exception observed was in the case of native RNase A, which is a poor substrate, yet it partially decreased the cross- linkage of '"I-labeled yeast cytochrome c. These findings suggest that the cross-linkage of labeled proteins mostly re- flects their binding to Ea.

Demonstration of a Functional &,.Protein Complex: Con- jugate Formation in Pulse-Chase Experiments-The above cross-linkage experiments suggest that protein substrates bind to Ea but do not necessarily indicate that binding is functional, i.e. that it leads to the conjugation of the protein with Ub. An approach to examining functional enzyme-substrate binding is the "isotope trapping" technique (22, 23). When a labeled substrate is first bound to its enzyme and then mixed with an excess of unlabeled substrate together with a second substrate that completes the reaction, part of labeled enzyme-bound substrate is converted to labeled product provided that product formation is faster than substrate


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I:[(;. :i ( / c / t ) . Cross-linking of lZ'II-labeled proteins to E:, suhunit . Cross-linking conditions were as described under ">laterials and Methods" except that E, (fraction D of phenyl-Sepharose chromatography, cf. Fig. 1) and bis(sulfosuccinimidy1)suherate were supplemented where indicated by + signs. The following IY5I-labeled proteins were used (numbers a t top): 1 , cytochrome c from S. cereuisae; 2, cytochrome c from horse heart; 3, lysozyme; 4, RNase A; 5,Ox-RNase; 6, MetO-RNase. All labeled proteins were supplemented a t 200,000 cpm (0.8- 1.3 pg). Positions of marker proteins (kDa) are indicated on the right side. Arrowhead, position of &-specific cross- link product. This is not much influenced by the protein, since the M, of all labeled proteins tested is in the range

FIG. 4 (center). Competition of cross-linking of 1z61-labeled yeast cytochrome c by various unlabeled proteins. All incubations contained 0.5 pg of "'I-labeled cytochrome c from S. cereuisae. Other cross-linking conditions were as described under "Materials and Methods." Lane I , without E2; lanes 2-7, with Ea; lanes 3-7, with the addition of 10 pg of the following unlabeled proteins: lane 3, cytochrome c from S. cereuisae; /one 4, cytochrome c from horse heart; lane 5, RNase A; lane 6, Ox-RNase; lane 7, MetO-RNase. Arrowhead, position of E,-specific cross-linking product.

FIG. 5 (right). Trapping of 1251-lysozyme bound to E3 for the formation of labeled conjugates. Pulse- chase experiments were conducted as described under "Materials and Methods," with the following variations: lanes 1-4, preincubation of "'I-lysozyme with E3; lanes 5-8, preincubation of '"I-lysozyme with E , and E*; lane I , chase mixture not supplemented lane 2, chase mixture lacking unlabeled lysozyme; lane 3, unlabeled lysozyme (60 pg) present in t,he pulse phase; lane 4, pulse-chase experiment. In lanes 5-8, 12sI-lysozyme was preincubated with a mixture of E,, E?, Me-Ub, and MgATP in a composition similar to that of the regular chase mixture but without unlabeled lysozyme. Lane 5, without further additions; lane 6, E, added in the chase phase but without unlabeled lysozyme; lane 7, unlabeled lysozyme (60 pg) added in the pulse incubation and then E, in the chase incubation; lane 8, E, and unlabeled lysozyme (60 pg) added in the chase incubation. All chase incubations were for 2 min. Contarn, contaminations in the preparation of "'I-lysozyme; Lys, '2sI-lysozyme.

of 12,000-14,500.

dissociation. In the experiment shown in Fig. 5 , '2sI-lysozyme (at a high specific radioactivity) was preincubated with ER (pulse), and then a 400-fold excess of unlabeled lysozyme was added together with E,, E2, Me-Ub, and MgATP, and incubation was continued for a short period of time (chase). Me- Ub was used instead of native Ub to prevent the formation of polyubiquitin chains (14), which would disperse any trapped radioactivity to more numerous products. Incorporation of ":'I-lysozyme radioactivity into conjugates was observed in the pulse-chase experiment (Fig. 5 , lane 4 ) in the regions expected from the control in which no unlabeled lysozyme was added (lane 2). Another control showed that isotope dilution by unlabeled lysozyme was sufficient to prevent the significant formation of labeled conjugates when unlabeled substrate was added prior to ER in the pulse phase (lane 3) . When '"I-lysozyme was preincubated with E2 and El and then Es was added at the chase phase, no significant formation of labeled conjugates was observed (Fig. 5 , lane 8). A parallel control to which unlabeled lysozyme was not added and ER was present only in the chase (lane 6) showed less conjugate formation than in the case of preincubation of Ea with lysozyme (lane 2). This is apparently due to the slow association of ":'I-lysozyme with ER (see below). These findings provide evidence for a functional enzyme-substrate complex and indicate that ER, but not E2, has the protein substrate binding site of the Ub ligation system.

Kinetics of Conjugate Formation and Es. Substrate Dissocia- tion-The time course of the formation of "'I-lysozyme-Me- Ub conjugates in the chase phase of the pulse-chase experiment is shown in Fig. 6 and the quantitation of the experiment in Fig. 7. It may be seen that the low-molecular weight conjugates 1-3 are formed in the first 10-30 s. Thereafter, radioactivity in bands 1-2 declines, whereas that in bands 4- 7 increases after a short initial lag. This suggests a precursor- product relationship between low- and high-molecular weight conjugates, though some low-molecular weight conjugates per- sist even after a prolonged chase incubation. I t should be further noted that the increase in radioactivity of high-molecular weight conjugates greatly exceeds the decrease in bands 1-2. The total amount of label in all conjugates increased until 2 min and approached a final level after 5-10 min of chase incubation (Fig. 7). In the experimental procedure employed (see "Materials and Methods"), mixing with excess unlabeled lysozyme was complete within 1 s of its addition. Therefore, the continuing accumulation of label in conjugates must originate from an &-bound pool of '2511-lysozyme. This implies that the dissociation of ER.12sI-lysozyme complex has to be slow relative to the rate of conjugate formation.

These assumptions were tested by the determination of the rate of dissociation of E,~.lZsI-lysozyme complex (Fig. 8). 9 - Lysozyme was preincubated with En, following which an excess of unlabeled lysozyme was added for various periods prior

11996

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FIG. 6. Time course of conjugate formation in the pulse- chase experiment.. Reaction conditions were as described under "Materials and Met.hods," except that the amount of '""Ilysozyme was increased 2-fold. The complete chase incubation was stopped after the following time periods: lane I , zero time (chase mixture not added); lane 2, 10 s; lane 3, 30 s; lane 4, 1 min; lane 5, 2 min; lane 6, 5 min; lane 7, 10 min. Numbers in the right margin indicate the different conjugates of ""I-lysozyme with Me-Ub (14) in order of increasing molecular size. Contam, contaminations in the preparation of ""I-lysozyme; Lys, "sl-lysozyme.

, I I 1 ,

1 2 3 4 5 6 7 8 9 1 0

Time (minutes)

FIG. 7. Quantitation of kinetics of formation of different conjugates in the pulse-chase experiment. The different bands of '"'I-lysozyme-Me-Ub conjugates shown in Fig. 6 were excised, and their radioactivity was estimated by gamma counting. Radioactivity of corresponding regions of the zero time sample was subtracted from all samples, and the results were expressed as the percentage of "'I- lysozyme radioactivity supplemented. Numbers in the figure indicate the correspondingly numbered conjugate bands in Fig. 6. Total, radioactivity in all conjugate bands 1-7.

to supplementation with a mixture of E, , EP, Me-Ub, and MgATP. The amount of labeled conjugates formed is a min- imal estimate of the amount of '2'II-lysozyme bound to Ea a t the time of addition of the EI/E2 mixture. I t may be seen that dissociation of "'I-lysozyme. ER complex is slow and has at least two components. About one-half of &-bound "'I-lysozyme dissociated with a half-time of about 10 s, whereas the

20 -

10 I , 1 1

1 2 3 4 5

Time (minutes 1 FIG. 8. Kinetics of dissociation of E3.'251-lysozyme com-

plex. Experimental conditions were as described under "Materials and Methods," except that unlabeled lysozyme (60 p g ) was added a t various time intervals before the addition of the chase mixture, which lacked unlabeled lysozyme. Following all additions, mixing was complete within 1 s. The zero time of the experiment was a sample in which unlabeled lysozyme was added together with the chase mixture (regular pulse-chase incubation). In all cases, incubation was continued for 5 min after the addition of El/& mixture. Following gel electrophoresis and radioautography, radioactivity in all conjugates was quantitated as described in Fig. 7. The results are expressed as the percentage of radioactivity in conjugates at time 0, which was 9500 cpm. Time denotes the interval between the additions of unlabeled lysozyme and of E1/E2 mixture.

I

I 2 4 6 8 10

Time (minutes)

FIG. 9. Time course of binding of '261-lysozyme to E3. Ex- perimental conditions were as described in "Materials and Methods" for the pulse-chase incubation except that '2'II-lysozyme was omitted from the pulse mixture. Following preincubation a t 37 "C for 5 min, "51-lysozyme was added, and incubation was continued for various time periods before the addition of the chase mixture. Chase incubation was for 5 min in all cases. Radioactivity incorporated into all conjugates was quantitated as described for Fig. 7. The results are expressed as the percentage of 12sI-lysozyme converted to conjugates. Time denotes the interval between the additions of 12sI-lysozyme and the chase mixture.

rest was released more slowly, tH - 2.7 min. This may be due either to heterogeneity of binding sites on Es or to a heterogeneity of '2'I-lysozyme molecules (see "Discussion").

The time course of the binding of '*'I-lysozyme to ER was examined in the experiment shown in Fig. 9. "'I-Lysozyme was incubated with Es for various periods before the supple-

Protein Binding Site of Ubiquitin Ligme 11997

mentation of the chase mixture, and the amount of labeled conjugates formed was estimated. The association of T - lysozyme with Es is also slow; half-maximal binding was a t around 1 min, and binding was complete only after 5 min.

Specificity of Binding of Protein Substrates to Es-Our next question was to what degree is the specificity of the Ub ligation system determined by the properties of the protein binding site of Ea. For this purpose, it was desirable to compare the affinity of various proteins to Es with their susceptibility to Ub ligation and degradation. We previously found that a free ru-NH, group of proteins is required for their degradation by the Ub system (4). However, not all proteins with free NH, termini are good substrates, and it is of interest to define which specific alterations render these proteins more suscep- tible to Ub ligation. It was previously noted (8) that RNase A (free (U-NH, group, poor substrate) is converted to a good substrate by performic acid oxidation (Ox-RNase). Oxidation of RNase with performic acid produces drastic alterations including cleavage of disulfide bonds, oxidation of methionine residues to methionine sulfone, and conversion of protein structure to a random coil (24). We now find that the specific oxidation of methionine residues to methionine sulfoxide (by photooxidation a t low pH) converts RNase to an even better substrate. The rate of ATP, Ub-dependent degradation of MetO-RNase is more than 2-fold faster than that of Ox- RNase (Fig. lo), and formation of high-molecular weight Ub conjugates of the former derivative was much more pro- nounced (Fig. 11). It is interesting to note that the rate of digestion of MetO-RNase by trypsin or chymotrypsin was 4- 5-fold less than that of Ox-RNase (data not shown), indicating a lesser degree of denaturation. Rather, some specific alteration produced by oxidation of methionine residues is appar-

FIG. 10. Influence of oxidation of methionine residues of RNase on i t s degrada t ion by the Ub system. Reaction mixtures contained in a volume of 200 pl: 50 mM Tris-HCI, pH 7.6,1 mM DTT, 5 mM MgCI,, 4 mM ATP, 12 pg U b , 40 pl fraction I1 from reticulocytes (approximately 1 mg of protein), and 4 pg (4-6 X lo5 cpm) of "'I- labeled proteins indicated in the figure. Incubation was a t 37 "C. At various times, aliquots of 50 pl were withdrawn and treated with 1 ml of 15% trichloroacetic acid. Release of acid-soluble radioactivity was determined as described previously (8). Parallel incubations were carried out without ATP, and ATP-dependent degradation was cal- culated by the difference. Above 90% of ATP-dependent degradation of all three substrates was also Ub-dependent.

1 2 3 4 5 6 -Origin

FIG. 11. Conjugation of oxidized derivatives of RNase with Me-Ub. Reaction mixtures contained in a volume of 20 pl: 40 mM Tris-HCI, pH 7.6, 1 mM DTT, 5 mM MgCI,, 2 mM ATP, 3 pg Me-Ub, 0.5 microunits of E,, 0.4 microunits of 172, 2.9 microunits of En, and 1 pg (approximately 300,000 cpm) of 12511-labeled proteins. Following incubation at 37 "C for 1 h, the reaction products were separated on a 12.5% SDS-polyacrylamide gel. Lunes 1-2, '"I-RNase A; lanes 3-4, 12sII-Ox-RNase; lanes 5-6, 12sII"etO-RNase; lanes I, 3, and 5, without El, E2, and En; lanes 2, 4, and 6, with E,, E?, and En.

ently recognized by the Ub ligation system. These protein substrates were used to characterize the

specificity of the binding site of Ea. As shown in Figs. 3 and 4, cross-linkage with Ea of various derivatives of RNase and cytochromes from different species was in correlation with their susceptibility to the action of the Ub system. We further examined these correlations, using the functional isotope trapping assay. When Es is incubated with 12sII-lysozyme and an unlabeled protein in the pulse phase of the pulse-chase experiment, decreased formation of '2'II-lysozyme-MeUb conjugates is due to competition on the E, binding site. Compe- tition at other sites of the ligase system (such as on E,-MeUb) is already maximal a t the chase phase, due to the presence of the large excess of unlabeled lysozyme. The results of such competition experiments are summarized in Table I. Yeast cytochrome c and yeast enolase (free NH, termini, good substrates for protein breakdown) effectively competed on the binding 12sI-lysozyme to Es, whereas equine cytochrome c and rabbit muscle enolase (N"-acetylated, not substrates) did not (Table I, experiment 1). Oxidation of methionine residues of lysozyme and RNase greatly increased their ability to inhibit Es (Table I, experiment 2). It should be noted, however, that native RNase also competed partially even though it is a poor substrate. This resembles a similar effect of this protein on Ea-substrate cross-linkage (Fig. 4). That the effects of all proteins tested were indeed due to competition for the E, binding site rather than the competence of the chase reaction was indicated by the observation that they had no significant influence when added at the chase phase of the pulse-chase incubation (data not shown).

From these results, it might be concluded that at least two structural features of proteins are recognized by the binding site of E3: a free a-NH2 group and oxidation of methionine residues. The question arises of whether a free (u-NH, group is obligatory for substrate binding. When amino groups of lysozyme were blocked by reductive methylation, its efficiency to compete on the binding of 12sI-lysozyme was greatly decreased (Table I, experiment 3). However, a derivative which was first methylated and then oxidized (MetO, Me-lysozyme) was a strong inhibitor of "'I-lysozyme binding even though

11998 Protein Binding Site of Ubiquitin Ligase TABLE I

Competition on binding of '25Z-lysozyme to E3 by natiue and modified proteins

Experimental conditions were as described under "Materials and Methods" for the pulse-chase experiment, except that the duration of the pulse incubation was 10 min. Unlabeled proteins were added before the pulse incubation at the amounts specified in the table. Radioactivity incorporated into all '251-lysozyme-Me-Ub conjugates was quantitated as described in Fig. 7. Results are expressed as the percentage of parallel control incubations to which no unlabeled protein was added in the pulse phase. These were 1.6-1.9% of I2'I-

lysozyme converted to conjugates in the different experiments. Protein added to pulse 1251-Lysozyme trapped

Clg % control Experiment 1

Cytochrome c, S. cereuisae 5 36.0 Cytochrome c, horse heart 5 95.0 Enolase, bakers' yeast 30 58.2 Enolase, rabbit muscle 30 104.0

Experiment 2 Lysozyme MetO-lysozyme RNase A Ox-RNase MetO-RNase

1.5 77.0 1.5 27.6 3 59.3 3 48.4 3 18.7

Experiment 3 Lysozyme 10 15.3 Me-Lysozyme 10 70.5 MetO, Me-lysozyme 10 0

its amino groups were blocked. It is concluded that proteins without a free a-NH2 group, or all amino groups, can bind to E3 provided that they contain another alteration which strongly increases their affinity to the protein binding site.

DISCUSSION

In this study we employed two independent methods, chemical cross-linkage and functional isotope trapping, to identify and characterize the protein substrate binding site of the ubiquitin-protein ligase system. Both methods indicated that Ea has the protein binding site, and both yielded essentially similar results with regard to the preferential binding of proteins that are good substrates for the Ub system.

The trapping of E3-bound '251-lysozyme for conjugate formation was allowed by the slow dissociation of E3. '251-lysozyme complex. The biphasic dissociation kinetics (Fig. 8) may indicate two types of binding sites on E3. Alternatively, it is possible that the population of lZ5I-labeled lysozyme molecules is heterogeneous. The latter possibility seems more likely, since radioiodination with the chloramine-T procedure causes the oxidation of some methionine residues to the sulfoxide derivatives (25). It is possible, therefore, that the more slowly dissociating component is due to a more extensively oxidized portion of 1251-lysozyme molecules, which have higher affinity for E3. It should be noted that unlabeled lysozyme used for chase experiments was subjected to a similar iodination procedure.

In view of the tight binding of '251-lysozyme to E3, it seems reasonable to assume that some endogenous reticulocyte protein substrates are also tightly bound to this enzyme. This may account for the observation that even the most highly purified preparations of EB are heavily contaminated by endogenous protein substrates for Ub conjugation (data not shown). The apparently slow binding of '251-lysozyme to E3 (Fig. 9) might be due to the slow dissociation of endogenous protein substrates, being replaced by the labeled protein.

Though E3 has not been purified to homogeneity, not much doubt remains about the identification of the 180-kDa protein

as its subunit, as shown by its coincidence with E3 activity in hydrophobic chromatography (Fig. l), second affinity chromatography (Fig. lA), gel filtration (Fig. 2), and hydroxylapatite chromatography (not shown). However, the subunit composition of E, is not clear. The native molecular size of E3 from rabbit reticulocytes had been previously estimated by gel filtration chromatography at around 300 kDa (8). The enzyme may be composed of two identical 180-kDa subunits or of one 180-kDa subunit with some lower-molecular weight subunits. I t also cannot be ruled out at present that the enzyme is composed of only one 180-kDa protein and that its higher apparent native size is due to its tightassociation with endogenous reticulocyte protein substrates. In any case, the 180-kDa protein contains the substrate binding site as shown by cross-linking experiments (Figs. 3 and 4 ) .

Examination of the binding of different proteins to E3 indicates that the specificity of the binding site may account for a part of the selectivity of the Ub ligation system. Proteins with free a-NH2 groups bind better to E3 than proteins with blocked a-NH2 groups, and proteins with oxidized methionine residues bind more tightly than their native counterparts. This correlates with the susceptibility of the above proteins to Ub conjugation and protein breakdown. However, in con- trast to the absolute requirement for a free a-NH2 group for Ub-dependent degradation (4), a methylated and oxidized derivative of lysozyme strongly competes on the binding site (Table I). In addition, native RNase A (free a-NH2, but bad substrate) inhibits significantly the binding of labeled substrates (Fig. 4 and Table I). It appears that proteins possessing only part of the structural requirements for Ub ligation may bind to E3 in an abortive complex. Further selectivity may be exerted at the stages of Ub conjugation, including ligation with the first Ub and the successive addition of multiple Ub molecules.

The observation that oxidation of methionine residues of RNase greatly increases its susceptibility to the Ub system (Figs. 10 and 11) raises the question whether a similar alteration may be a physiological signal for protein degradation. Oxidative damage of methionine residues in proteins probably occurs in cells, and an enzyme system was described which specifically reduces methionine sulfoxide residues in proteins (26). The oxidation of a single histidine residue has been implicated in the inactivation and breakdown of glutamine synthetase (27, 28). In the present study, the effect of the oxidation of all methionine residues in a model protein was examined, and further studies with more physiological substrates are required to assess the possible significance of this observation.

In addition to the protein binding site reported in this study, it appears reasonable to assume that E3 contains some other sites necessary for its action in Ub-protein ligation. The observation that E3 can be isolated by affinity chromatography on Ub-Sepharose (8) indicates that this enzyme has Ub binding site(s). E3 has to interact with E,-Ub for Ub-protein ligation to occur. In addition, it is possible that some Ub- protein conjugates are also tightly bound to E3. Since ligation with multiple molecules of Ub is observed in spite of substrate excess, it has been suggested that the reaction is either processive or has a strong preference for proteins conjugated to Ub (29). In the present investigation, the continuing utili- zation of intermediates in the presence of high amounts of unlabeled lysozyme (Fig. 6) suggests that both the bound protein and its conjugates with Ub have very slow rates of dissociation. The affinity of E3 to Ub might be due to the presence of specific sites for the above complexes which contain Ub moieties. Further investigation is needed to define other binding sites of EB, involved in other stages of the action of the Ub-protein ligase systems.


Acknowledgments-We thank Dr. Irwin A. Rose for discussions, 13. Ciechanover, A., Elias, S., Heller, H., and Hershko, A. (1982) J. suggestions, and much help. We acknowledge the assistance of Sarah Bwl. Chem. 257,2537-2542 ~ l i ~ ~ at the initial stages of this work, We also thank Clara Segal 14. Hershko, A., and Heller, H. (1985) Biochem. Biophys. Res. Com- and Judith Hershko for excellent technical assistance. 15. Bohlen. P.. Stein. S.. Dairman. W.. and Udenfriend. S. (1973)

mun. 128, 1079-1086

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the protein substrate binding site of the ubiquitin-protein ligase

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