THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. No. Issue of ... · THE JOURNAL OF BIOLOGICAL CHEMISTRY 0...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biologi, lnc.

Vol. 267, No. 23, Issue of August 15, PP. 16403-16411,1992 Printed in U. S. A.

Inhibition of Ubiquitin-Protein Ligase (E3) by Mono- and Bifunctional Phenylarsenoxides EVIDENCE FOR ESSENTIAL VICINAL THIOLS AND A PROXIMAL NUCLEOPHILE*

(Received for publication, January 24,1992)

Erica S . BerlethS, Eileen M. Kasperek, Susan P. Grill, Julie A. Braunscheidel, Lynne A. Graziani, and Cecile M. Pickart8 From the Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, New York 14214

Trivalent arsenoxides bind to vicinal thiol groups of proteins. We showed previously that the simplest trivalent arsenoxide, inorganic arsenite, inhibits ubiquitin-dependent protein degradation in rabbit reticulocyte lysate (Klemperer, N. S. , and Pickart, C. M. (1989) J. Biol. Chem. 264, 19245-19242). We now show that, relative to arsenite, phenylarsenoxides are 10-165-fold more potent inhibitors of protein degradation in the same system for inhibition by p- aminophenylarsenoxide was 3.5-20 PM, depending on the substrate). In the ubiquitin-dependent proteolytic pathway, covalent ligation of ubiquitin to protein substrates targets the latter for degradation. In certain cases, specificity in ubiquitin-substrate conjugation de- pends critically upon the properties of ubiquitin-protein ligase or E3. Among other effects, p-aminophenylarsenoxide decreased the steady-state level of ubiquitinated human a-lactalbumin; this is a substrate which is acted upon directly by ubiquitin-protein ligase-a (E3-a). This finding suggests that phenylarsenoxides (unlike arsenite) inhibit E3. Several other lines of evidence confirm this conclusion. 1) A complex of E3-a and the 14-kDa ubiquitin-conjugating (E2) isozyme binds to phenylarsenoxide-Sepharose resin, with the E3 component of the complex mediating binding. 2) p-Aminophenylarsenoxide inhibited isolated E3

-50 PM); inhibition was readily reversed by addition of dithiothreitol (which contains a competing vicinal thiol group), but not by j3-mercaptoethylamine (a monothiol). 3) A bifunctional phenylarsenoxide (bromoacetylaminophenylarsenoxide) rapidly and irreversibly inactivated E3; bromoacetyl aniline, which lacks an arsenoxide moiety, did not inhibit E3. These results suggest that E3 possesses essential vicinal thiol groups and that there is a reactive nucleophile proximal to the vicinal thiol site. The bifunctional phenylarsenoxide should be a useful tool for probing the rela-

*This work was supported by Grant DMB89-04984 from the National Science Foundation, including a Research Experiences for Undergraduates Supplement. Some of this work has been described in a preliminary communication (Berleth, E., Kasperek, E., Grill, S., Braunscheidel, J., and Pickart, C. (1991) FASEB J. 5 , A1179 (Abstr. 4685)). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by a predoctoral fellowship from the Woodburn Foun- dation during part of this project. Present address: Roswell Park Cancer Institute, Buffalo, NY 14263.

8 To whom correspondence should be addressed: Dept. of Biochem- istry, 140 Farber Hall, SUNY, Buffalo, NY 14214. Tel.: 716-831-2355; Fax: 716-831-2725.

tionship between structure and function in E3. As expected from prior results with arsenite, p- aminophenylarsenoxide was also a potent inhibitor of the turnover of ubiquitin-(human) a-lactalbumin conjugates.

A major protein degradation pathway in eukaryotic cells utilizes the highly conserved protein ubiquitin as a covalent proteolytic signal (reviewed in Refs. 1, 2). In this pathway ubiquitination renders the target protein susceptible to a specific ATP-dependent protease, and ubiquitin is regener- ated during degradation by one or more specific isopeptidases. Target protein ubiquitination occurs in two or three steps, depending on the identity of the substrate (3). Initially, ATP hydrolysis is required to link the COOH-terminal Gly residue of ubiquitin to a thiol group of El.’ Ubiquitin is then transferred to a thiol group of an E2. The E2-Ub thiol ester is the proximal donor of ubiquitin to a Lys €-amino group of the target protein, or of a previously conjugated ubiquitin molecule. A t least seven E2 proteins exist in mammalian cells and most are capable of ubiquitinating certain proteins under defined conditions (reviewed in Ref. 2). However, only one isozyme, E214K, is so far known to mediate formation of conjugates which are efficiently degraded in vitro (4-6). In the synthesis of such conjugates, transfer of ubiquitin from E 2 1 4 ~ to the substrate is catalyzed by a third enzyme, ubiquitin-protein ligase (E3) . Much of the documented substrate selectivity of the ubiquitin proteolytic system resides in the properties of E3 ( 1 ) .

In the yeast Saccharomyces cereuisiae, the turnover rates of recombinant proteins differing only in their amino-terminal residues correlate with the identities of the target proteins’ amino-terminal residues, suggesting that the conjugative ma- chinery recognizes the substrate’s amino terminus (7). By using dipeptides and amino acid derivatives to inhibit E3- catalyzed ubiquitination, it was shown that E3 is the N-end-

The abbreviations used are: El, ubiquitin activating enzyme; E2, ubiquitin conjugating enzyme (subscript denotes molecular mass in kDa); E3, ubiquitin-protein ligase; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography; HPLC, high performance liquid chromatography; DTNB, dithionitrobenzoate; DTT, dithiothreitol; PAsO, phenylarsenoxide; NPAsO, p-amino phenylarsenoxide; BrAcNPAsO, p-[(bro- moacety1)-amino]phenylarsenoxide; BrAcAn, bromoacetyl aniline; H-LA, human a-lactalbumin; B-LA, bovine a-lactalbumin; rcmBSA, reduced and carboxymethylated bovine serum albumin; P-LGB, p - lactoglobulin; PAsO-Sepharose, phenylarsenoxide-Sepharose; Ph- Sepharose, phenyl-Sepharose; MOPS, 4-morpholinepropanesulfonic acid; HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid.

16403

16404 Vicinal Thiols in Ubiquitin-Protein Ligase

recognizing conjugative enzyme (8, 9). Three classes of E3 substrates have been defined (7-9). Type I substrates have basic amino-terminal residues (His, Arg, Lys); Type 11 substrates have bulky hydrophobic amino-terminal residues (Phe, Tyr, Trp, Leu); and Type I11 substrates have small uncharged amino-terminal residues (Ser, Thr). Substrates bearing acidic amino termini (Glu, Asp) are post-translationally arginylated by Arg aminoacyl-tRNA protein transferase in order to become Type I substrates (10). Two different molecular species ofE3 have been identified in rabbit reticulocyte lysate through the use of immobilized substrate affinity columns. Reticulo- cyte E3-a appears to contain, on one 180-kDa polypeptide, distinct sites which bind Type I and I1 substrates ( 1 1 ) . This latter inference is consistent with the properties of an E3-a homolog recently cloned from S. cereukiue (12). E3-@ exhibits preference for Type I11 substrates, although it is not clear that it recognizes substrates uia their amino termini (13). E3- a and E3-@ share several properties (see Ref. 13), including a large native molecular mass (2250 kDa) and a requirement for E2 in ubiquitination.

We showed previously that arsenite, a trivalent arsenoxide which induces the stress response, inhibits ubiquitin-dependent protein degradation in rabbit reticulocyte lysate (see Ref. 14). Targets of arsenite included Arg aminoacyl-tRNA transferase and an unidentified component involved in conjugate turnover. Trivalent arsenoxides bind to vicinal thiol groups on proteins (15 ) . In an effort to understand the catalytic roles of vicinal thiol groups in ubiquitin-dependent proteolysis, we synthesized mono- and bifunctional phenylarsenoxides (15) and examined their inhibitory effects on ubiquitin-mediated degradation. In addition to the sites of inhibition expected from our previous work, there was an unexpected effect on the ubiquitination of substrates which are acted upon by E3- a. This effect is described in the present report. We show that a monofunctional phenylarsenoxide is a reversible inhibitor of E3-a, while a bifunctional arsenoxide is an irreversible inactivator of the same enzyme. A phenylarsenoxide-sepharose column was among the tools used to demonstrate the E3- phenylarsenoxide interaction.

EXPERIMENTAL PROCEDURES~

Unless specified otherwise, materials were identical to those described previously (14). In some cases, reticulocytes for the preparation of ubiquitin-depleted lysate (fraction 11) were obtained commercially (Green Hectares, Oregon, WI). Immediately prior to experiments involving arsenoxides, DTT (0.2 mM) was removed from fraction I1 by centrifugal gel filtration through Sephadex G-25 (16). ~-[2,3-~H]Arg (53 Ci/mmol) for synthesis of [3H]Arg-rcmBSA was obtained from Du Pont-New England Nuclear. The following proteins were radioiodinated by the chloramine-T method (17) to specific radioactivities of 106-106 cpmlpg: H-LA, B-LA, 8-LGB, rcmBSA (18), RNase S-protein, and ubiquitin. SDS-PAGE was carried out in mini- gels by the discontinuous slab procedure of Laemmli (19). Several experiments were done with commercially obtained PAsO (Aldrich); stock solutions were prepared in dimethyl sulfoxide and diluted using aqueous Tris-HC1 (10 mM, 24% base). Affinity-purified polyclonal antibodies raised against a truncated form of the S. cerevisiae RAD6 protein (anti-rad6I4’ antibodies of Ref. 20) were a gift of P. Sung and L. Prakash (University of Rochester). Ubiquitin aldehyde preparations were provided by K. Wilkinson (Emory University) or R. Cohen (UCLA). El and E2,,, were purified to electrophoretic homogeneity (5); their concentrations were determined as described previously (4). Ubiquitination of free lysine was monitored as described (4). Other protein concentrations were determined using Bio-Rad dye reagent

Portions of this paper (including Table I and part of “Experimen- tal Procedures”) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

concentrate, with bovine serum albumin as standard. P-LGB-Sepha- rose was prepared, and used to purify E3-a from reticulocyte fraction 11, exactly as described previously (11).

E3 Assay-Assays (10 pl, pH 7.3, 37 “C) contained 50 mM Tris- HCl (24% base), 5 mM MgC12, 10 mM phosphocreatine, 2 mM ATP, 0.6 unit/ml each of creatine phosphokinase and pyrophosphatase, lZ5I-ubiquitin (2 or 5 pM; -8,000 cpm/pmol), 100 nM El , and 200 nM E2,4K. One of the following E3-a substrates was added H-LA (1 mg/ ml), S-LGB (2 mg/ml), or oxidized RNase (0.1 mg/ml). Assays were initiated by adding 0.5-1.5 microunits of E3 (unit as defined in Ref. a), quenched after 3-5 min with 10 pl of SDS-PAGE sample buffer, and then heated to 100 “C for 1 min. An aliquot (15 p1) was electro- phoresed (12.5% gel); the gel was dried and autoradiographed. The region of the lane containing ubiquitinated proteins was excised and counted. For the blank, E3 was omitted. Appropriate controls indicated that ubiquitin conjugates were formed linearly with time. Where E3 was preincubated with or without phenylarsenoxides at 37 “C, the buffer contained 10-50 mM Tris-HC1(24% base) and 0.1 mM EDTA.

RESULTS

The degradative substrates utilized in this work can be classified based on the pathway by which each is ubiquitinated (Table 11). All require E l and E214K (3, 4). In the actual conjugative step, H-LA (Type I) and @-LGB (Type 11) are directly acted upon by E3-a ( 1 1 ) . Ubiquitination of RNase S- protein (Type I11 substrate) is apparently catalyzed predom- inantly by E3-/3 (13). B-LA and rcmBSA have NH2-terminal residues with acidic side chains and become Type I substrates of E3-a following NH2-terminal arginylation catalyzed by Arg aminoacyl-tRNA transferase (10).

The reagents used in this work are shown in Scheme I. PAsO and NPAsO are

O = A s - o - R R = H PAsO R = NH, NPAsO R = NHC(=O)CH2Br BrAcNPAsO

H- 0 “ R ’ R’ = NHC(=O)CH2Br BrAcAn

monofunctional reagents which should inhibit solely by binding to vicinal thiols (Equation 1).

BrAcNPAsO is (potentially) a bifunctional reagent; its arsenoxide moiety is comparably reactive to that of NPAsO, but

TABLE I1 Inhibition of ubiquitin-dependent protein degradation by

phenylursenoxides Assays of degradation of the indicated radioiodinated substrates

were carried out in reticulocyte fraction 11 in the presence of 9.4 p~ ubiquitin, as described under “Experimental Procedures.” Values of KO, and nH were determined graphically using Hill plots. See also Fig. 1.

Reagent Substrate NHI terminus (type) RH KO.,, MM

PAsO rcmBSA Asp (I)“ 2.0 25*,‘ NPAsO rcmBSA Asp (I)” 2.0 20 NPAsO H-LA Lys (I) 1.8 3.5‘ BrAcNPAsO H-LA Lys (I) 2.1 3.8 NPAsO B-LA Asp (I)’ 1.8 3.5 NPAsO 8-LGB Leu (11) 2.0 19 PAsO 8-LGB Leu (11) 1.9 22 NPAsO RNase S-protein Ser (111) 1.8 3.5‘ Undergoes NHp-terminal arginylation prior to ubiquitination.

* Independent of whether fraction I1 was added last to the assay versus assays in which fraction I1 was preincubated with arsenoxide for 10 min at 37 “C, without MgATP, prior to initiating the assay.

e Maximum inhibition was 88% (legend, Fig. 1B).

Vicinal Thiols in Ubiquitin-Protein Ligase 16405

[NPaSO]. uM

FIG. 1. Inhibition by NPAeO of ubiquitin-dependent degradation and effect on steady-state conjugate level. Degrada- tion assays (filled symbols), and measurement of steady-state conjugate level (open symbols), were carried out as described under “Ex- perimental Procedures.” All values are expressed relative to controls lacking NPAsO. Panel A , radioiodinated rcmBSA (e, 0) and [3H] rcmBSA (V). For lZSI-rcmBSA, incubations (2 h) were initiated by adding fraction I1 protein (3.2 mg/ml): degradation (e); steady-state conjugate level (0). Control values: 23.1% soluble (degradation); 4479 counts/4 min (conjugate level, determined at 1 h). Data from two separate experiments are combined. For [3H]rcmBSA degradation (V), incubations (90 min) were initiated by addition of ubiquitin and MgATP (see “Experimental Procedures”). Usually 10% of the [3H]rcmBSA was degraded in the control incubation lacking NPAs0. These data were corrected assuming that the counts made soluble in the presence of 100 p~ NPAsO were equal to a blank lacking ubiquitin; this was verified in preliminary controls. Results of three experiments are combined in the figure (mean f S.D.). The line was calculated assuming n~ = 2.0 and Ko.5 = 20 p ~ . Panel B, radioiodinated H-LA (V, V) and RNase S-protein (e, 0). For H-LA, incubations (15 min) were initiated by adding fraction I1 protein (1.4 mg/ ml). All values except that for 1 p~ NPAsO are averages of duplicates which agreed within 520%. Control values: 36.6% soluble (V, degradation); 5424 counts/2 min (V, conjugate level, determined at 10 min). For RNase S-protein, incubations (1.8 mg/ml fraction I1 protein; 30-40 min) were initiated by adding labeled substrate. Control values: 31% soluble (0, degradation); 4400 counts/3 min (0, conjugate level; determined at 10 or 15 min). Results of five separate experiments are combined (mean * S.D.), except for 1, 50, and 100 p~ which are averages of duplicates. The line was calculated assuming n~ = 1.8 and K0.6 = 3.5 p ~ , and defining the observed maximal inhibition (88%) as 100%.

the alkyl halide moiety of BrAcNPAsO can irreversibly al- kylate nucleophilic amino acid side chains. The extent to which irreversible inactivation by BrAcNPAsO involves at- tack by a nucleophile within an arsenoxide-binding site may be evaluated through the use of BrAcAn, which lacks the arsenoxide moiety.* NPAsO, BrAcNPAsO, and BrAcAn were originally synthesized and characterized by Stevenson and co-workers (15).

Reversible Degradative Inhibition by Monofunctional Phen- ylarsenoxides-ubiquitin-mediated degradation of each of the above-mentioned substrates was inhibited by the monofunctional reagents PAsO and NPAsO, with inhibition constants which ranged from about 4 to 20 p ~ , and which were not significantly different for PAsO uersus NPAsO (Table 11). Without exception, inhibition was positively cooperative ( nH = 2; Fig. 1 and Table 11).

In all cases, premixing of phenylarsenoxide with excess DTT (relative to [arsenoxide]) completely prevented inhibition (data not shown). Moreover, addition of excess DTT to NPAsO-inhibited assays almost completely reversed inhibition in all cases (the rate after DTT addition ranged from 75

Second-order rate constants for the reaction of the thiol group of free cysteine with the alkyl halide moiety: 19 M”min” (BrAcAn) and 33 M-lmin” (BrAcNPAsO). Conditions: 50 mM MOPS, 1 mM EDTA, 50% (v/v) methanol, 37 “C (J. Li and C. Pickart, unpublished experiments).

2 0

UM NPASO

10 20 30 40 I, mln

FIG. 2. Inhibition of turnover of ubiquitinated H-LA. Pulse incubations contained 50 mM Tris-HC1 (20% base), 5 mM MgCL, 0.1 mM ATP, 0.6 unit/ml pyrophosphatase, 9.4 p~ Ub, -6 pg/ml Iz5I-H- LA, and 3.6 mg/ml fraction I1 protein. After 5 min at 37 “C, aliquots of the pulse were diluted 1.2-fold into chase incubations containing final concentrations oE 700 pg/ml unlabeled iodinated H-LA (14), 2 mM ATP, 5 mM phosphocreatine, and 0.3 unit/ml creatine phosphokinase, and other components as indicated below (final volume, 80 pl; dilution represented time zero in the chase). At the times indicated on the abscissa, 13-pl aliquots were withdrawn and quenched with 7 pl of 2 X concentrated SDS-PAGE sample buffer. Conjugate level was determined as described (14), with counting for 4 min; there were 4000 cpm at time zero). Chase conditions: no addition (0); 5 p~ NPAsO (A); 10 p~ NPAsO (0); 50 p~ NPAsO (A). Inset, relative first-order rate constants are plotted as a function of [NPAsO] in the chase; results of two experiments are combined. The line was calculated assuming hyperbolic inhibition, with KO.5 = 5 p ~ .

to 100% of the control lacking arsenoxide, data not shown). In contrast to the effect of the dithiol DTT, cysteine (2 mM) did not prevent or reverse inhibition caused by 50 p~ NPAsO (experiments with lZ5I-RNase S-protein, data not shown). The failure to achieve reversal with monothiols indicates that phenylarsenoxides do not inhibit by binding to protein monothiol groups. The ability of a dithiol (DTT) to reverse inhibition is consistent with the hypothesis that phenylarsenoxides inhibit by binding to vicinal thiol groups of one or more ubiquitin pathway components (15, 21, 22).

Effect of NPAsO on Ubiquitin Conjugate Turnover-Based on our previous work with inorganic arsenite, the simplest arsenoxide, we anticipated that monofunctional phenylarsenoxides would reversibly inhibit two steps in ubiquitin-mediated degradation: 1) arginylation catalyzed by Arg aminoacyl-tRNA transferase; and 2) turnover of ubiquitinated forms of some substrates (14). Phenylarsenoxides are indeed potent inhibitors of the transferase; these results will be described el~ewhere.~ The effect of phenylarsenoxides on ubiquitin conjugate turnover was investigated only with H-LA. Ubiquitin- ated forms of H-LA were generated during a brief pulse with lZ5I-H-LA, and conjugate turnover was monitored kNPAsO during a chase with excess unlabeled substrate, in the presence of MgATP. Isopeptidase and protease activities, which both contribute to conjugate disappearance during this type of chase (14), were not separately monitored. NPAsO inhibited turnover of conjugated H-LA with KO., = 5 p~ (inset, Fig. 2); this effect is probably the primary cause of inhibition of overall degradation of this substrate = 3.5 p ~ ; filled triangles, Fig. 1B). Addition of 1.5 mM DTT to a chase at 11 min fully reversed inhibition of ubiquitinated H-LA turnover (experiment with 31 p~ NPAsO, data not shown).

Apparent Inhibition by NPAsO of Ubiquitin-Protein Con- jugation-Since NPAsO strongly inhibited turnover of ubi-

5E. Berleth, J. Li, J. Braunscheidel, and C. Pickart, manuscript submitted.


A 1 2 3 4 5 6 7 8 9

B

6 4 1 3 5 7 9 II 13 15 17 19

C .( c

1 3 5 7 9 I I 13 15 I7 19 21 23

-14K

D I 3 5 7 9 I t 13 15 17 19 21

+ + -14K

FIG. 3. An E~-E!&X complex binds to PAsO-Sepharose. Panel A , small scale experiments. All operations were carried out at 5 “C. Fraction I1 protein (5 mg, 280 pl) was rotated with 100 pl of PAsO-Sepharose in a buffer of 20 mM Tris-HC1(24% base). After 60 min, the resin was pelleted by centrifugation in a microfuge. The supernatant (unbound fraction) was removed, and the resin was washed with 1 ml of the same Tris buffer. The washed resin was rotated with 200 pl of elution buffer (20 mM Tris-HC1, 0.5 mg/ml ovalbumin, 20 mM DTT) for 30 min; after pelleting the resin, the supernatant (DTT eluate) was removed. Lanes 1-6, conjugation assays (autoradiograph). Determinations of the level of ubiquitinated “‘1-H-LA (“Experimental procedures”) were carried out on 3.3-pl aliquots of the unbound fraction and DTT eluate, or 2.6 pl of untreated fraction 11. Lane I , blank (no enzyme source); lane 2, untreated fraction 11; lanes 3 and 4, unbound fraction; lanes 5 and 6, DTT eluate. Purified El, 100 nM, was added to the assays in lanes 4 and 6. H-LA, 1251-labeled substrate; cont, radiolabeled contaminant in substrate. The bands migrating above the contaminants are ubiquitin- H-LA conjugates. Lanes 7-9, Western blot. Proportional aliquots of untreated fraction I1 (lane 7), the unbound fraction (lane S), and the DTT eluate (lane 9) were analyzed using anti-RAD6 antibodies (“Experimental Procedures”). The positions of E 2 1 4 ~ and E 2 2 5 ~ are indicated. The identity of the high molecular weight immunoreactive band is unknown. Panels B and C, large scale experiment. Preparation of the DTT eluate, and its fractionation on a glycerol gradient, are described under “Experimental Procedures”. Panel B, conjugation assays. Assays employing aliquots (5 pl) of the indicated gradient fractions were carried out as described for panel A, except that El (100 nM) and ubiquitin aldehyde (1 p ~ ) were included in all assays. The profiles for fractions 21 and 23 (identical to 19) have been omitted. Panel C, Western blot. The same fractions as in panel B were analyzed using anti-RAD6 antibodies. An identical result was obtained when NPAsO was included in the glycerol gradient buffer

quitinated H-LA, moderate concentrations of NPAsO should increase the steady-state level of ubiquitin-H-LA conjugates (14). Instead, concentrations of NPAsO above 5 PM decreased the steady-state level of ubiquitin-”’I-H-LA conjugates (open triangles, Fig. 1B). This latter result suggests that NPAsO inhibits E l , E214K, or E3-a, all of which are required for ubiquitination of H-LA (3, 4, 8, 11). Such inhibition was unexpected, since inorganic arsenite did not inhibit ubiquitin- protein conjugation (14).

With H-LA, inhibition of ubiquitination was weak (Ko.5 -25 p ~ ) relative to inhibition of overall degradation (open versus filled triangles, Fig. 1B). This behavior reflects the strong inhibition of conjugate turnover seen with this substrate (inset, Fig. 2), which is probably the predominant cause of degradative inhibition at low concentrations of NPAsO (above, Ref. 14); moreover, inhibition of conjugate turnover may partially mask inhibition of conjugate formation, since steady-state conjugate levels are seen in fraction I1 (“Discus- sion”). With the substrate rcmBSA, however, an inhibitory effect on steady-state conjugate level quantitatively paralleled inhibition of degradation -20 pM; open versus filled circles, Fig. LA). RcmBSA is a substrate whose ubiquitination is transferase dependent, and NPAsO inhibits the transferase (above). However, in the experiment shown by the filled triangles in Fig. lA, we used rcmBSA containing [3H]Arg at its NH, terminus as the degradative substrate; since arginylated rcmBSA is the product of the transferase reaction, any effect of NPAsO on transferase activity is irrelevant. Results obtained with [“HIArg-rcmBSA and 1251-(Asp)rcmBSA were not significantly different (triangles versus filled circles, Fig. LA). Thus, NPAsO inhibits a step in rcmBSA degradation other than that catalyzed by the transferase. We showed previously that arsenite inhibits turnover of ubiquitin- rcmBSA conjugates only very weakly (14). Thus, it is likely that, as seen with H-LA, NPAsO inhibits ubiquitination of (arginylated) rcmBSA. In support of the idea that NPAsO inhibits one of the three enzymes required generally for ubiquitination (El, E214K, E3), the decrease in the steady-state conjugate level was half-maximal for both substrates at a similar NPAsO concentration, -20-25 PM.

E3-a Binds to Phenylarsenoxide-Sephurose-To demonstrate a direct interaction between one or more conjugative enzymes and phenylarsenoxide, we passed fraction I1 proteins over a PAsO-Sepharose column (“Experimental Procedures”). The column was functionally similar to that described recently by Zhou et al. (23) but was prepared by a different method. Zhou et al. used N-hydroxysuccinimide and dicyclo- hexylcarbodiimide to link the amino group of NPAsO to a resin bearing carboxymethyl groups (23). We reacted the alkyl halide of BrAcNPAsO with the thiol of P-mercaptoethylamine, and reacted the amino group of the resulting conjugate with activated CH-Sepharose.

As shown in Fig. 3A (lanes 2 versus 3 and 4 ) , the unbound fraction resulting from this procedure showed reduced activity in ubiquitination of 1251-H-LA, a substrate of E3-a. Elution of the phenylarsenoxide column with DTT yielded a fraction with activity in ubiquitination of Iz5I-H-LA (Fig. 3A, lane 5 ) . Stimulation of this activity by added El (Fig. 3A, lanes 5 versus 6) indicated that El did not bind to the column with

(“Experimental Procedures”; data not shown). Panel D, control West- ern blot. Partially purified E~, ,K was sedimented (“Experimental Procedures”). In panel D, only the region of the blot containing E211~ is shown; no other immunoreactive bands were present. The gradient yielded 22 fractions (rather than 23). The arrows in panels E-D indicate the peak fractions of catalase (250 kDa, fraction 8) and carbonic anhydrase (30 kDa, fraction 18), as determined on a separate gradient.


high efficiency. The other two enzymes required for ubiquitination of H-LA, E2,,K and E3-a, apparently bound to the column and were released by DTT. Western blot analysis with antibodies that cross-react with several reticulocyte E2s (20) confirmed that E214K was present in the DTT eluate (Fig. 3A, lanes 9 versus 7, and 8). Visual inspection of the results of two such experiments suggested that 30-50% of the in fraction I1 bound to the PAsO-Sepharose column. Since the unbound fraction resulting from these manipulations had almost no activity in ubiquitination of 1251-H-LA (Fig. 3A and data not shown), the resin apparently bound most of the E3- a in fraction 11.

The following observations indicate that there is a high degree of specificity in the binding of E214~ and E3-a to PAsO-Sepharose. 1) El did not bind efficiently (above). 2) The El-supplemented eluate was not active in degradation of l2’1-H-LA (data not shown), indicating that the conjugate- specific protease did not bind. (However, recombination of the bound and unbound fractions fully reconstituted degradation; data not shown.) 3) E225K, which is also detected by these antibodies (20), bound very poorly (Fig. 3A, lanes 8 versus 9). 4 ) Ovalbumin exhibited negligible binding (data not shown). 5) When fraction I1 was passed over phenyl- Sepharose (lacking the arsenoxide moiety), the unbound fraction was fully active in ubiquitination of ’251-H-LA, and there was no detectable E3-a activity in the DTT eluate (data not shown).

An E2. E3 complex has been described previously, although it was not shown whether the complex involved a specific E2 isozyme (24). To address whether E3-a and E214K bound to PAsO-Sepharose as a complex, we fractionated the DTT eluate on a glycerol gradient (24). If they are uncomplexed, E3-a (250 kDa) and E214K (-30 kDa) should be base line- resolved. Instead, when the DTT eluate from PAsO-Sepha- rose was fractionated, ‘251-H-LA conjugation assays (done with added El, but without added E214K) revealed a peak of activity sedimenting at -200 kDa (Fig. 3B). Based on the known enzymatic requirements for ubiquitination of H-LA, this peak must contain E3-a and E214K. Consistent with this expectation, Western blot analysis showed that a fraction of the E214K in the DTT eluate also sedimented at -200 kDa (Fig. 3C). In contrast, when E 2 1 4 ~ was run alone, all of it sedimented at -30 kDa (Fig. 3 0 ) . We conclude that at least a fraction of the E214~ in the DTT eluate exists in a high molecular weight complex with E3-a. This complex may correspond to the E2. E3 complex described previously (24).

Which component mediates binding of this complex to PAsO-Sepharose? The following observations indicate that E3-a mediates binding. 1) In assays of free lysine conjugation catalyzed by purified Ez14K, NPAsO, 50-200 p ~ , caused only slight and variable inhibition (520%, data not shown). 2)

did not bind to PAsO-Sepharose in the absence of E3 (“Experimental Procedures”). 3) E3 did bind to PAsO-Seph- arose in the absence of E214~ (“Experimental Procedures”). 4 ) The bifunctional reagent BrAcNPAs0 inactivated partially purified E3-a irreversibly and completely, in a manner dependent upon its arsenoxide moiety (below). The same reagent (150 p ~ ) inactivated E214K by only 12% (not shown). We conclude that when fraction IJ is applied to PAsO-Seph- arose, E 2 1 4 ~ binds primarily as part of an E2. E3 complex. However, a large amount of uncomplexed was apparently present in the DTT eluate (Fig. 3C). This may reflect partial dissociation of the E2eE3 complex during the time (-20 h) required for concentrating, dialyzing, and fraction- ating the eluate. Overall, the results shown in Fig. 3 suggest that phenylarsenoxides bind to E3-a and that this interaction

causes the inhibition of ubiquitin-protein conjugation documented in Fig. 1 (open symbols) and below.

Mono- and Bifunctional Phenylarsenoxides Inhibit Partially Purified E3”We used PAsO-Sepharose chromatography as the final step in a partial purification of E3 (“Experimental Procedures”). The resulting DTT eluate contained a promi- nent band migrating on SDS-PAGE a t about 200 kDa (Fig. 4, lane 3) . This band may represent, at least in part, the known E3 subunit (-180 kDa (11)). Many other proteins were also present in the DTT eluate. Degradative reconstitution assays with ’*‘I-H-LA showed that this E3 preparation contained E3-a (assays as in Ref. 5 , data not shown). Since we do not know whether this preparation contains E3+, it will be referred to as an “E3 preparation.” The standard E3 assay employs labeled ubiquitin and unlabeled substrate (“Experi- mental Procedures”), and proteins contaminating the E3 preparation are among those conjugated (below). Thus, it is possible that E3-P (if present) makes a contribution to the “E3 activities” reported below. The method utilized for E3 purification assured that our preparations were devoid of E2230K, which is also inhibited by phenylarsenoxides.3

Varying the concentration of E214K in the assay showed a saturating dependence of rate on E214K concentration, with K, = -200 nM (data not shown). We were unable to determine a K,,, for conjugative substrate because of competition from endogenous substrates contaminating the E3 preparation. The concentrations of exogenous substrates used in standard assays typically gave a 2-fold stimulation; the rate did not increase further when the exogenous substrate concentration was increased.

NPAsO inhibited partially purified E3, with KO, = 53 ~ L M (Fig. 5). DTT, but not P-mercaptoethylamine, reversed inhibition (Fig. 5A). E3-a purified using a substrate affinity column (P-LGB-Sepharose (11)) was comparably sensitive to NPAsO (reversible inhibition with KO, -40 p ~ ; data not shown), indicating that use of PAsO-Sepharose in E3 purification did not select for a subpopulation of arsenoxide-sensitive E3 molecules. The dependence of E3 inhibition on [NPAsO] was evaluated at different E 2 1 4 ~ concentrations. The value of for NPAsO increased by 20% when [ E 2 1 4 ~ ] was increased from 50 to 200 nM (two experiments, data not shown). If E214K and NPAsO bound competitively to E3, KO.’ would be expected to increase by 60% (cf. K, = 200 nM, above). Therefore, E2,4K and NPAsO probably do not bind competitively to E3. This conclusion is consistent with the finding that an E3 ‘E214K complex binds to PAsO-Sepharose (above) and with the observation that inclusion of NPAsO (0.3 mM) in the glycerol gradient buffer did not decrease the

1 2 3

Y I

- 0

FIG. 4. Partial purification of E3 on PAsO-Sepharose (Coo- massie Blue-stained SDS-polyacrylamide gel). Purification was carried out as described under “Experimental Procedures.” Lane 1, high salt fraction from Q-Sepharose (30 pg of protein); lane 2, unbound fraction (30 pg); lane 3, DTT eluate (12 pg of protein, of which about 40% is carrier ovalbumin, designated oualb). The arrowhead denotes the possible E3 subunit. The molecular masses of standard proteins are shown on the right.


1004 ,I” 1

uM NPArO

FIG. 5. Panel A , reversible inactivation of E3 by NPAsO. Partially purified E3 (“Experimental Procedures”) was preincubated without (m) or with (0,0, V) 375 p~ NPAsO prior to diluting (at t = 0) 2.5- fold into a tube containing all other assay components; the exogenous substrate was H-LA. (The degree of inhibition was similar whether E3 was preincubated with NPAsO, or was the last addition to the assay; data not shown.) At the time indicated by the arrow, aliquots of the assay (containing 150 p~ NPAsO) were diluted into tubes containing concentrated DTT (V) or P-mercaptoethylamine (MEA, 0), to give a final concentration of 10 mM with negligible dilution of the assay. Aliquots of the different assays were quenched and analyzed at the indicated times (“Experimental Procedures”). Panel B, concentration dependence. Initial rates in E3 assays are expressed relative to a control lacking NPAsO. The abscissa shows the [NPAsO] in the assay. The l ine was calculated assuming hyperbolic inhibition with KO.’ = 53 p~ and maximal inhibition of 83%. Each point includes data from several independent experiments (mean -C S.D., n = 4-6).

fraction of E214K sedimenting at 250 kDa (“Experimental Procedures,” legend, Fig. 3C).

In contrast to the reversible effect of NPAsO, the bifunctional reagent BrAcNPAs0 irreversibly inactivated E3: when 170 p~ BrAcNPAsO was preincubated with E3 for 1 min prior to initiating the assay, adding excess DTT at t = 1.5 min in the assay did not restore activity (open triangles, Fig. 6A) . However, DTT afforded 80% protection when present during the preincubation between E3 and BrAcNPAsO (filled versus open circles, Fig. 6A). Thus, there is very rapid reaction of an E3 nucleophile with BrAcNPAsO, with concomitant E3 inactivation. The degree of irreversible inactivation occurring during a 1-min preincubation followed a cooperative concentration dependence, with -120 p~ (nH = 2, Fig. 6B) . In Fig. 6B, excess DTT was added to the assay at zero time (together with all other assay components); therefore, E3 is the inactivated species. In other qualitative experiments (not shown), we found that irreversible inactivation was time dependent; this accounts for the difference in the degree of inactivation caused by 170 p~ BrAcNPAs0 in Fig. 6A (2.5 min elapsed before DTT addition) uersus Fig. 6B (1 min elapsed before DTT addition).

When E3 was preincubated for 1 min with 300 p~ BrAcAn, followed by assay in the presence of excess DTT, there was no inactivation (not shown). This contrasts with the 92%

t, rnin [BrAcNPAsO]. uM

FIG. 6. Panel A, irreversible inactivation of E3 by BrAcNPAsO. Final conditions were those of the standard E3 assay, with oxidized RNase as substrate. The assay volume was 22 pl. At the indicated times, aliquots (5 pl) were withdrawn, quenched, and analyzed as described under “Experimental Procedures.” Filled circles, E3 was preincubated without inhibitor for 1 min prior to initiating the assay with a mixture of all other components (time zero). Open circles, BrAcNPAsO (0.17 mM) was incubated with DTT (1.8 mM) for 1 min; E3 was added and the assay was initiated after 1 min. Filled triangles, E3 was preincubated with 0.17 mM BrAcNPAsO for 1 min prior to initiating the assay. Open triangles, as for filled triangles, except that DTT (1.8 mM) was added to the assay at 1.5 min. Panel B, concentration dependence. Filled circles, partially purified E3 was preincubated with the concentration of BrAcNPAsO indicated on the abscissa for 1 min (volume = 6 pl). Assays were initiated by adding 4 pI of a mixture of the other assay components and DTT (5 mM final). Open circles, the indicated concentration of BrAcNPAsO was preincubated with 5 mM DTT for 4 min, E3 was added, and the assay was initiated after 3 more min. Data from two independent experiments are included in the figure. The line was calculated assuming nH = 2.1 and

= 117 pM. Ub, ubiquitin.

(irreversible) inactivation occurring during a comparable incubation with BrAcNPAsO (Fig. 6B) . Therefore, irreversible inactivation by BrAcNPAsO is strictly dependent upon its arsenoxide moiety. Inclusion of saturating NPAsO (8-10 times together with 375 p~ BrAcAn did not bring about irreversible inactivation (data not shown). This result argues against the possibility that a conformation change induced by arsenoxide binding enhances the reactivity of a nucleophile that is distant from the arsenoxide-binding site. We conclude that a nucleophile proximal to the arsenoxide binding site of E3 reacts with the alkyl halide moiety of bound BrAcNPAsO.

As required based upon its ability to inactivate E3 irreversibly, BrAcNPAsO is an irreversible inactivator of ubiquitin- dependent degradation of “’I-H-LA and ‘“’I-rcmBSA. When fraction I1 was incubated with 30 p~ BrAcNPAsO for 12 min (37 “C), ubiquitin-dependent degradation of H-LA was abolished; incubation with 50 p~ BrAcNPAsO for 30 min abolished degradation of rcmBSA (data not shown). These find- ings do not exclude the possibility that additional arsenoxide- sensitive components of the ubiquitin-dependent proteolytic pathway are also irreversibly affected by this reagent. Indeed, BrAcNPAsO is an irreversible inactivator of Arg aminoacyl- tRNA protein transfera~e.~ Other potential irreversible effects of this reagent, including effects on conjugate turnover, have not yet been evaluated.

The effect of phenylarsenoxides on E3-P was not directly examined. However, NPAsO decreased the steady-state level of ubiquitinated ”‘I-RNase-S-protein in fraction I1 (KO, - 10 p ~ , open circles, Fig. 1B). While this effect is most simply explained if NPAsO inhibits E3$, further work will be required to confirm this inference.

DISCUSSION

Inhibition of Ubiquitin-dependent Protein Degradation by Phenylarsenoxides-Based on earlier results obtained with


inorganic arsenite (14), we expected phenylarsenoxides to inhibit ubiquitin-dependent degradation. The present results show that phenylarsenoxides are more potent degradative inhibitors than is arsenite, by factors ranging from 10- to 165- fold, depending on the substrate (compare Table I1 with Table I of Ref. 14). Phenylarsenoxides are among the most potent inhibitors of the ubiquitin proteolytic pathway yet known. They are comparable to hemin (KO.5 = 25 pM) as inhibitors of rcmBSA degradation (25). The specific El inhibitor adenosyl- phospho-ubiquitinol is a more potent inhibitor of 0-LGB degradation than are phenylarsenoxides (K0.5 < 1 p~ for the ubiquitinol (26)). However, this reagent does not inhibit degradation of rcmBSA (26). Phenylarsenoxides, in contrast, similarly inhibit degradation of rcmBSA and 0-LGB (K0.5 -20 p ~ ) . The facile reversal by DTT of (monofunctional) phenylarsenoxide inhibition should increase the utility of these reagents as inhibitors of the ubiquitin pathway.

In two cases, the inhibited steps identified in the present work were those predicted from earlier results with arsenite. As inhibitors of conjugate turnover and of Arg aminoacyl- tRNA protein transferase, phenylarsenoxides are distin- guished from arsenite primarily by affinity. Thus, turnover of ubiquitinated H-LA is inhibited by NPAsO with K0.5 = 5 p~ (Fig. 2) versus 190 p~ for arsenite (14). While in the present study we did not separately evaluate effects on deconjugation versus conjugate proteolysis, we showed previously that inorganic arsenite similarly inhibited de-ubiquitination and degradation of ubiquitin-H-LA conjugates (14). As seen previously with arsenite, the sensitivity of conjugate turnover to phenylarsenoxides presumably varies for different substrates; otherwise inhibition of overall degradation would follow Ko.5 -5 p~ in all cases (cf. Fig. 2). The transferase is also more potently inhibited by phenylarsenoxides than by arsenite?

Phenylarsenoxides Inhibit E3”The finding that phenylarsenoxides depress the steady-state level of ubiquitin-protein conjugates (Fig. 1) was unexpected (below). In contrast to effects on the transferase and on turnover, which vary quali- tatively (transferase) and quantitatively (turnover) between substrates, phenylarsenoxides decreased the levels of conjugates of three different substrates, with a relatively similar concentration dependence (K0.5 -10-25 pM, Fig. 1). Pheny- larsenoxides decrease steady-state conjugate level because they inhibit E3. This conclusion is based on several lines of evidence. First, a physical interaction between E3 and phenylarsenoxide was revealed by the binding of E3 to PAsO- Sepharose, either alone (“Experimental Procedures”) or in a complex with E214~ (Fig. 3). Second, inhibition by NPAsO was demonstrated in direct assays of isolated E3 (Fig. 5). Third, BrAcNPAsO inactivated E3 rapidly and irreversibly in a manner strictly dependent upon the presence of the arsenoxide moiety of this bifunctional reagent (Fig. 6 and “Results”). Finally, mono- and bifunctional phenylarsenoxides do not inhibit E214K, nor is there any evidence that they interact with El (“Results”). Our results show unambiguously that phenylarsenoxides inhibit E3-a; they suggest that E 3 4 may be inhibited also, although this inference remains to be confirmed. The failure to detect E3 inhibition in prior experiments with millimolar concentrations of inorganic arsenite (14) presumably reflects much weaker binding of arsenite, as seen for two other arsenoxide-sensitive steps (above). We estimate that the phenyl substituent must potentiate arsenoxide binding to E3 by at least 100-fold.

With the monofunctional reagent NPAsO, addition of a dithiol, but not a monothiol, rapidly reversed E3 inhibition (Fig. 5A). This characteristic, which was seen in all cases of inhibition described here and previously (14), is consistent

with inhibition deriving from phenylarsenoxide binding to vicinal thiol groups (15, 21, 22). Arguments that these are vicinal thiol groups of enzymes, as opposed to protein substrates, have been summarized previously (14). For E3, preincubation of enzyme and BrAcNPAsO abolished activity in a subsequent assay carried out at high [DTT] (below); moreover, E3 bound to phenylarsenoxide immobilized on a column. Thus, with E3 it is virtually certain that the vicinal thiols reside on the enzyme.

BrAcNPAsO, but not BrAcAn, inactivated E3 irreversibly, indicating that there is a reactive nucleophile proximal to the vicinal thiol site(s) of E3. (These data do not address the number of vicinal thiol sites/molecule of E3; for simplicity, we will refer to a single site.) The proximal nucleophile may play an essential role in catalysis; alternatively, it may be a non-essential residue, but addition of the bulky bifunctional reagent may hinder access to other essential residues in the site. BrAcNPAsO is the only irreversible inactivator of E3 yet identified. Among other potential uses (below), this reagent may provide a simple means to determine whether conjugative reactions in crude systems are E3-mediated. At present, we do not understand why BrAcNPAsO inactivates E3 completely, while NPAsO does not (Fig. 5B versus 6B). Nor do we understand the basis of the positive cooperativity seen with BrAcNPAsO (Fig. 6B).

Since irreversible inactivation of E3 by BrAcNPAsO re- quires initial binding via the arsenoxide moiety of this reagent, reversible inhibition by NPAsO and irreversible inactivation by BrAcNPAsO are expected to follow a closely similar concentration dependence. The apparent 2-fold greater potency exhibited by NPAsO (Fig. 5B versus Fig. 6B) is probably a consequence of the time dependence of irreversible inactivation (“Results”). The true K 0 . 5 for irreversible inactivation would be determined by measuring the concentration dependence of the inactivation rate constant. Quantitative experiments of this type were precluded by the very rapid inactivation seen at high concentrations of BrAcNPAsO.

Reversible inhibition of isolated E3 was half-maximal at -50 ~ L M NPAsO (Fig. 5B). In reticulocyte fraction 11, the decrease in the steady-state level of ubiquitin conjugates of rcmBSA and H-LA was half-maximal at -25 p~ NPAsO (Fig. 1). Further work will be required to determine why these values differ. The level of conjugates in fraction I1 was meas- ured at a subsaturating concentration of labeled test substrate (“Experimental Procedures”), but in the presence of abundant endogenous substrates whose fractional saturations are unknown. In addition, the conjugate level in fraction I1 reflects a steady-state of E3-dependent conjugate formation and isopeptidase/protease-dependent conjugate turnover; the latter steps may also be arsenoxide-sensitive (cf. Fig. 2). These considerations indicate that only a qualitative K0.5 value for phenylarsenoxide binding to E3 can be determined in reticulocyte lysate. Assays with isolated E3 were done in the presence of a saturating concentration of exogenous substrate; if substrate and phenylarsenoxides were to bind competitively, the observed value of KO would underestimate the true affinity of E3 for phenylarsenoxide. Determining whether phenylarsenoxides and protein substrates bind to the same or independent sites awaits the availability of better preparations of E3 (below).

These current data do not address the catalytic roles of the vicinal thiols, or the associated nucleophile, except in a neg- ative way: kinetic results, and the ability of an E3. E214K complex to bind to PAsO-Sepharose, suggest that the vicinal thiols are localized in a site distinct from that which interacts specifically with E214~. One possibility, at present speculative,


is that the vicinal thiols function to facilitate ubiquitin transfer between bound E214K and bound substrate; this role could involve formation of one or more E3-ubiquitin thiol ester intermediates. The functions of vicinal thiol groups in the pyruvate dehydrogenase complex (15), and in lecithin choles- terol acyl transferase (22)) provide enzymatic precedent for this type of mechanism.

As noted above, future studies on the mechanistic role of vicinal thiols in E3 catalysis must employ purer E3 preparations than those used here. [14C]BrAcNPAs0 has been used to identify a His residue of lipoamide dehydrogenase that is proximal to the lipoamide cofactor of lipoate acetyltransferase (21). This same reagent should provide a straightforward route to identification of the nucleophile within the vicinal thiol site of E3, provided adequate quantities of enzyme can be obtained. The kind of phenylarsenoxide "affinity" resin described here and by others (23) should be helpful in attain- ing both of these objectives. The phenylarsenoxide resin will not provide a single-step purification of E3, due both to nonspecific binding of other proteins ( c f . Fig. 4) and to binding of other arsenoxide-sensitive pathway components. However, the latter problem can be avoided by a judicious choice of early purification steps ("Experimental Procedures"). PAsO- Sepharose is simple to use, has a high capacity, and provides fairly good recovery of activity. This resin should thus be a useful addition to the battery of methods currently available for E3 purification.

Acknowledgments-We are grateful to Edward Turos for advice and assistance in the syntheses of NPAsO and BrAcNPAsO. We thank K. J. Stevenson for communicating the HPLC protocol used in arsenoxide characterization and for helpful discussions. Arash Emami provided assistance in preliminary studies on inhibition by PAsO. We are grateful to Jun Li for determining the chemical

reactivities of the bromoacetyl moieties of BrAcAn and BrAcNPAsO and to Louise Prakash and Patrick Sung for providing antibodies.

1. 2.

3.

4. 5. 6.

8. 7.

9.

11. 10.

12. 13. 14.

15.

16. 17.

18. 19. 20.

21.

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28,6035-6041

SUPPLEMENTARY MATERIAL to:

lnhibitionolubiquitln-protcin ligase(E3) by mono-and bifunctional phenylarsenox1dps:~vidrnee for eSIentisl vicind thiols. and P proximal nucleophile

Erica S. Berleth, Elleen M. Ka5perek. Susan P. Crdl. Julie A. Braunseheldel, Lynne A Grazlan~, and C e d e M. Plckart

by

EXPERIMENTAL PROCEDURES Tahle I


M e ~ s ~ r e r n e n l orsleBdy-Itate de#rsdation rate and ubiquilin conjugate level (rsdioiadinstrd rub- rtmter). Degrrdanon was monmured as the formatwn of rrichloroacetw aad-soluble radioactwny (pH 7.3. 3 7 T ) SO mM Tnr-HCI (24% hare). 5 mM MgC12. 2 mM ATP, 10 mM creatlne phosphate. 0 6 ti/ml each

wbwate protem (k /K conditions [14]). S m n the malo' fractlon of the degradatwe actwity was depend- of c r e a m phosphokmrse and m r g a n x pyrophorphatasc, Y.4 uM ubiqumn. and ~ Id cpmI2S U I of labeled

ent upon added ub&tit(70% for H-LA. B-LA and RNAre.S-protetn. 80% for L)-LGB. and 80.95% for rcmBSA). rates were not corrected for uhiqultin-independent degradauon. In one cxperiment, NPArO mhthtlcd H-LA degrrdation in the absence of added ubquitm with K,, - 5 uM. eiosely Simdar 10 the value (3.5 uM) obtamcd in the presence of added ublqumn (Table 11). Som: i i t h s 'ublqultln-depcndent" brcak- duwn probably ~nvolver trace ublquitm ~n fracuon 11.

analyw of altquors removed from actual degradauon assays (14). For assays with 'l'I-RNAre S-prorein. t ihlqwinated forms of rrdiomdmaled substrates were wrualmed and quanttlared by SDS PAGE

fraction 11 piutem ( I0 mg/ml) war premeubaled w~ th ub tqum aldehyde (1.6 uM) for 5 min (25°C) prmr to mwatmg assays This treatment had no effect on de radation rates. hut increased steady-state canlugate levels sl~ghtly. Steady-state levels of ubiquitinated 'iSI-rcmBSA were determined aflcr SDS qucnchmg by mmunoprcdpltat,on wnh ant~-ubtquam antbodles (14. 30).

tography on Q-Sepharore fast flow (Pharmaeia-LKB) as described ( 5 ) . E3 artiuily was defmcd as ublquilin. Parlial pwincalion of W on PAsO.Sephsm%e. Fracuon I 1 war subjected to anion exchange chroma-

conlugarlng a m v q dependent on addttmn of purlfled E2 E3 actwity war generally 3- to 5-fold higher ~n the fracuon eluting between 0 3 and 0.5 M KCi. relatwe I:& fraetlon eluting at 50.3 M KCI. The hlgh-salt fractwn, whlrh contains a low level of E2,, (5. 6) and a low level of EZYK (31'. was somclmes used for E3 purlfmwon an PAsO-Scpharore (below). bore frequently. we generate a hlgh salt fraction that war devomd of EZ,,& and EZ1= as follows. A 0.30% ammonium sulfate cut war made from fraction 11. The 0.30% ammomum sulfate &mon mntamr E3 but lacks E2r (6, 31). The 0-30% cut w a then applxd to Q-Sepha- rae (as ahove)

The hlgh-salt fraction was dmlyred m o buffer A (0.2 M Tr~a-HCI [24% base]. 0 1 mM EDTA) a n - lainmg 0.2 mM D l T and appllcd to PAsO-Sepharou (10-25 mg prown/ml rem) ~n 5-10 ahquuts (at I-mm mtervalr). Unbound prolcmr were collected on ice ~ n t o a lube eontaming D l T . The loaded column was washed wlrh 5-10 vols of buffer A (wlthout DTT). Bound proteins were eluted over 10 min wtth 5 vols of huffer A eonrainmg ovalbumln (tamer, 0 4 mg/ml) and DTT (5-10 mM) When the eluate war to be used in arrenoxldc lnhibitlon studler. 11 was repeatedly concentrated (Centricon-IO. Amleon) and diluted with buffer A to make [Dm-0.2 mM Purified E3 was stored tn small ahquotr at -b0'. Freermg and thawing of elu- ates whlch lacked D l T altogether inactivated €3.' E3 specific a c t ~ t y in the final DTT eluate. calculated bared on the roncenrrarmn of non-ovalbumm protem. war generally 3- to 4-fold higher than in !he startlng

0-Sepharore htgh-salt fraction. Usually about 80% of the E3 actwnty war recovered. wtth abut half of thts

unbound E3 muid be recovered by re-appiying the flow-through to the column'). Comparable results were in the D l T eluate, and the rcmamder m the unbound fracrlon (due to column overloading, most of the

sulfate fractmn (above). Hence E3 can bmd to PAsO-Sepharaie independently of E21,1(. For the prcpara. obtained when startmg from high-salt fractions derived from total fraction 11. or from the 0.30% ammonium

tmn shown ~n Fig. 4. ammonium sulfate fractionation was nM done.

t m 11. #E fraction 1s devotd of E3 (6). The (dialyzed) proteins were fractmated on 0-Sepharore; pro. €2 dors not bind 10 PAsO.Sephamsc. We prepared a 36.80% ammomum sulfate cut from frac-

teins eluting between 0 25 and 0.36 M KC1 were pooled, concentrated, and dialyzed into buffer A (above) mntamng 5 mM cyrteme. The presence of E2 $r th!: fraction was verlfied by thml ester assay wnh '*I- ublqultin ( 5 ) . When this fraction was applied: As0 Sepharore as described above. the ~ C I U I I S ofthiol ester assays showed that all detectable E2,,K was I" the unbund fractlon (data not shown). Cysteine does not block rpeche binding to PAsO-Sepharosc?

gradient frartiomtlon. Operatiom were carried out using a barx buffer of 20 mM Trir-HCI (24% base) and Lary-%*le sppliention or rraetion I I to PAsO.Sephamse, prrpantion or DlT eluate. and @ycrml

0 1 mM EDTA. Fraermn I1 (8.4 ml. 5 mgjml protein) was applied 10 a 3-ml column of PAsO4epharore ~n ths buffer. The column was washed with I5 ml of buffer. and then eluted with 12 ml of buffer containmg DIT (20 mM) and ovalbumm (carrier. 0.5 mgjml). The D I T eluate was repeatedly concentrated and diluted with the initial buffer (Centncon-IO. Amicon), to yield 0.4 ml eluate mntainmg 0.25 mM D I T . Duplicate aiquols (0.2 ml each) of thls eluate were applted to tubes contaming 4 4-ml glycerol gradtents whleh had been prepared exactly as dcscrlbed p r c v ~ ~ s l y (24). except that in one of the two gradient buffers, 1 mM DTT was replaced by a mixture of cysteine ( 5 mM) and NPAsO (0.3 mM). Two additional gradients were prepared. one for fractlonauon of a mixture of carbonic anhydrase and calala~e ( s m standards). and the other for fracuonatmn of 0.1 ml of a crude preparation of E2,4K. The four rubes were centrifuged smulta- neoudy. ab dercrlhed (24). and fractmnr (-0.2 ml) were collected from each. Aliquots (10 ui) of ailernale fraettom were mtxed with I O UI of SDS-PAGE sample buffer. and 15 UI of each sample war run on a 12 5% acrylamlde gel The SIX standard gel was stained with Coomawe blue. Protetnr in the other three geis were

20, except that alkalme phosphatare-conlugated goat anti-rabbit IgG [BiwRadl was used for colormetrlc transferred to lmmobdon (Mtllnpore), and blots were lmmunostalned wlth an1i-rad6~~~0antlbodler (as tn Ref

delectlon). Assays ofublqumn conjugation were carried out on aliquotr ( 5 ul) of certam fracllons. Final condmonr ( I O "1, 37OC) were the same as for assay of E3. except that: the substrate war '%H.LA (IO' cpm/asray). uhqumn (Y.4 uM) was unlabeled. E2 war pmltted, and El war omltted except where Indieat- ed Assays were quenched after 5 mm wlth 10 ul% SDS PAGE sample buffer. followed by electrophoresis and autoradiography.

E. Berleth and C. Pickart, unpublished experiments.

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