Alterations in components of the ubiquitin-protein ligase system following maturation of reticulocytes to erythrocytes

Vol. 145, No. 2, 1987 June 15, 1987

AND BIOPHYSICAL

BIOCHEMICAL

RESEARCH COMMUNICATIONS Pages 658-665

ALTERATIONSIN COMPONENTS OF THE UBIQUITIN-PROTEINLIGASE SYSTEM FOLLOWING MATURATIONOF RETICULOCYTES TO ERYTHROCYTES Osnat Raviv,

Hannah Keller,

and Avram Hershko

Unit of Biochemistry, Faculty of Medicine, Technion - Israel Institute of Technology, Haifa 31096, Israel Received April

20, 1987

Summary: Previous studies have shown that the activity of the ubiquitin-mediated proteolytic system declines markedly following reticulocyte maturation, but the specific alterations responsible for this phenomenonhave not bee72$.efined. We find that the rate of ATP-dependent degradation of I-albumin is reduced 20-fold in lysates of rabbit erythrocytes, as compared to reticulocyte lysates. The activity of the proteolytic system in erythrocyte extracts can be restored by supplementation with components of the ubiquitin-protein ligase system purified from reticulocytes by affinity chromatography. These components are the ubiquitincarrier protein E2, the activity of which is nearly completely absent, and the ligase E3, the activity of which is partially reduced in erythrocytes. Erythrocyte extracts contain other ligases which attach a single, or a few ubiquitin molecules to proteins; these products are different from the multi-ubiquitin derivatives which are formed by the ligase system of protein breakdown. Mature red cells may thus serve to distinguish between different ubiquitin-protein ligase systems with presumably different o 1987 Academic Press, Inc. functions. Studies on the mode of action of an ATP-dependent proteolytic system from reticulocytes of ubiquitin

in protein

of the intermediary pathway.

have led to the discovery

of the role

breakdown and to the elucidation

reactions

of the ubiquitin-mediated

In this pathway, proteins

of many proteolytic

destined for degradation are

first conjugated to the polypeptide ubiquitin and are then degraded by enzyme(s) that specifically attack ubiquitin-conjugated proteins (for reviews, activity

see

of this

1,2).

The reason for the exceptionally

system in reticulocytes

been suggested that one of its the removal of mitochondrial

main functions

Abbreviations: DTT, dithiothreitol; serum albumin.

Inc. reserved.

in this cells

of reticulocytes

(3).

is

In mature

' 25,-,SA , ' 25I-labeled

0006-291X/87$1.50 0 1987 by Academic Press, of reproduction in any form

It has

and someother unnecessary proteins

in the process of the maturation

Cowright All rights

is not clear.

high

658

bovine

Vol. 145, No. 2, 1987

red cells,

BIOCHEMICAL

most of the activity

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

of the proteolytic

system is lost

concomitant with the complete loss of protein

(4-7),

synthesis.

The question arises as to the cause of the loss of the activity of the proteolytic system in mature erythrocytes. Since the system is CcmFosed of multiple enzyme components, the loss of any component would result

in the loss of ubiquitin-dependent

proteolysis.

Previous in the conjugation

studies showed that three enzymes are involved of ubiquitin 8,9),

to proteins:

ubiquitin-carrier

E3 (10,12).

a ubiquitin-activating proteins

The intermediary

tin-conjugated

proteins

(E2's, refs.

reactions

enzyme (El, ref.

in the degradation of ubiqui-

have not yet been elucidated,

that three enzyme components and ATP are involved investigation

of the levels

tes has not been carried components persist levels

of ubiquitin,

(14,15).

now report

of the different

out, but it

to protein

Systematic

components in erythrocy-

Erythrocytes

and are the preferred

contain high

source for its

purification

also contain the ubiquitin-activating

comparable to those in reticulocytes

that the loss of proteolysis

to the decline of E2, and to a partial ubiquitin-protein

but it appears

(13).

is known that at least some

in mature red cells.

Mature red cells

enzyme El in levels

and the ligase

10,ll)

ligase activities

breakdown, persist

(9).

in mature red cells limitation

of E

3'

which are apparently

We is due

Someother unrelated

in mature red cells.

METHODS Lysates from rabbit reticulocytes were prepared as described previously (10). Erythrocgte lysates were prepared as follows: All operations were at O-4 C. Blood from healthy rabbits was collected on ice in the presence of heparin. The cells were centrifuged (1000 x g, IOmin) and washed 4 times with 4-5 volumes of ice-cold phosphate-buffered saline. The huffy coat was throughly removed by aspiration in each wash. Contamination of erythrocytes by reticulocytes was less than ./+%,as estimated by staining with Basic Blue 24 (Sigma). The cells were lysed by the addition of 1.5 volumes of ImM DTT . The preparation was allowed to stay on ice for 30 min, following which it was centrifuged at 82,000 x g for 2 hours, in a Beckman SW-27 rotor. The supernatant was collected by careful aspiration and was stored at -7O'C in small samples. Fraction II (a crude, ubiquitin-free fraction) was prepared from lysates of erythrocytes or reticulocytes by chromatography on DEAEcellulose as described (10). Affinity chromatography of Fraction II on ubiquitin-Sepharose was carried out as described previously (10). Assay of protein breakdown. The reaction mixture contained in a volume of 50 ~1: 50 mMTris-HCl (pH 7.6), 5 mMMgC12, 3 mMDTT, 0.5 mMATP 10 &I phosphocreatine, 0.6 unit of creatine phosphokinase, 1 ug of 1251-BSA ($10 x IO&pm) and 2 ug of tRNA from calf liver (Boehringer). tRNA was included because it stimulates the degradation of 1251-albumin (16). Lysates from erythrocytes or reticulocytes, and other enzyme components were supplemented as described in the legends to Figures. Following incubation at 37OC for 2 hours, the release of trichloroacetic acid659

Vol. 145, No. 2, 1987

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

soluble material was determined as described (10). Parallel incubations were carried out without ATP, phosphocreatine and creatine phosphokinase With and ATP-dependent proteolysis was computed by the difference. 1251-BSA as substrate, ATP-independent proteolysis was always less than 10% of ATP-dependent protein breakdown, in agreement with previous results (17). Determination of activities of E2 and E3. These were assayed by the quantitative determination of the conjugation of 125 I-ubiquitin, as described previously (IO), under conditions in which the other two components of the ligase system are supplemented in excess. The reaction mixture contained in a volume of 50 ~1: 50 mMTris-HCl (pH 7.6), 5 mM MgC12, 2 mMATP, 2 mMDTT, 0.04 units of yeast inqs3anic pyrophosphatas (Sigma), 20 ug oxidized ribonuclease and 50 pmol I-ubiquitin (2-5x10 z CPm). E2 activity was determined in the presence of 1.2 uunits of El and 1.1 uunits of E3, while E3 activity was determined with the same amount of El and 0.8 uunits of E2 (see ref. 10 for preparation of these enzymes and the definition of unit of activity). Following incubation at 37OC for 30 min, the radioactivity of ubiquitin-protein conjugates formed was determined by adsorption onto a mixture of amino- and cation-exchange resins, as described (10). RESULTSAND DISCUSSION In the experiment shown in Fig. 1, the activity proteolytic

system in lysates

in reticulocytes.

A drastic

of rabbit

erythrocytes

decline in the activity

system is seen.

The residual

throcyte

was 44% of that in reticulocytes.

lysates

agreement with our earlier the activity

of the proteolytic

in intact

obervations

These findings cells

in eryare in

on the decline of

reticulocyte

of Speiser and Etlinger

in

(7).

restored in erythrocyte fraction

was compared with that

of ATP-dependent proteolysis

system following

(5) and with similar lysates

Speiser and Etlinger ubiquitin

observations

of the proteolytic

maturation cell-free

activity

of the ATP-dependent

also reported that ATP-dependent proteolysis extracts

is

by the supplementation of a crude

from reticulocytes

(7).

Since erythrocytes

contain

large amounts of active ubiquitin (14), it seemedpossible that some other factor, contained in the samecrude fraction, is missing from erythrocytes. However, in numerous attempts we could not reproduce the above results. We therefore examined which other components of the ubiquitin proteolytic system decline following reticulocyte maturation. The three enzymes of the ubiquitin-protein ligase system can be isolated by affinity

chromatography on ubiquitin-Sepharose,

and are partially

separated from each other by elution under specific conditions (9,lO). In the experiment illustrated in Fig. 2, we tested the restoration of protein breakdown in erythrocyte lysates by fractions purified from reticulocytes

by affinity

DTT eluate of the affinity protein breakdown partially.

chromatography.

The supplementation of the

procedure, which contains mainly E2' restored The addition

of pH 9 eluate,

which contains

Vol. 145, No. 2, 1987

BIOCHEMICAL

60-

AND BIOPHYSICAL

Retie.

i601

Gate

04ol-

I

RESEARCH COMMUNICATIONS

0

DTT

Eluate

Erythr.

f2 k!J

20

10 Lysate

30

(pl)

LA

2

w

Affinity

4

6

Fraction

8

Added

10

12

(pg

of protein)

14

Fig. 1. Comparison of ATP-dependentproteolytic activities of lysates from,e&hrocytes and reticuIoc,ytes. ATP-dependent proteolysis of "'I-BSA was determined as described under I'Methods" in the presence of varying amountsof lysates from rabbit erythrocytes ( M ) or reticulocytes (O-0 ). Fig. 2. Restoration of protein breakdownin erythrocyte extracts by fractions isolated from reticulp&,es by affinity chromatograph,y.ATP-dependentbreakdownof '"'I-BSA was determined as described under "Methods", in the presence of 30~1 lysate from erythrocytes and the indicated amounts of DTTeluate (D-0 ) or pH 9 eluate (0-0 ) from affinity

chromatography

E3 in addition

to E2

in erythrocyte

extracts

of Fraction

II

from reticulocytes

completely reconstituted

(IO),

(Fig.

protein

(10).

breakdown

2).

Since the DTT- and pH 9-affinity

eluates contain several other proteins with high affinity for ubiquitin in addition to E2 and E 3 (10,12), it was desirable to examine further whether these two enzymes are indeed missing in erythrocytes. This was done by chromatography of the pH 9 eluate by gel filtration and examination of the coincidence of activity

which restores

with activities E2 exists

protein

of E2 and E3 (Fig.

breakdown in erythrocyte 3).

lysates

It was found previously

that

in two forms with apparent molecular sizes of 25,000 and 250,000,

while E3 elutes in one peak in coincidence with the higher molecular weight form of E2 (10). As shown in Fig. 3, activity which stimulates protein

breakdown eluted in two peaks, a minor peak coinciding

with low-

molecular-weight E2, and a major peak which is coincident with the combined peak of E3 and high-molecular-weight E2. These results suggest that the decline of E2 and E3 cause the loss of activity of the proteolytic system in mature erythrocytes. The results in Figs. 2 and 3 also indicate that while the loss of E2 is essentially 661

complete, that of E3 is only

BIOCHEMICAL

Vol. 145, No. 2, 1987

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

I 20

30

40

Fraction

50

60

Number

3. Co-elution of activities which stimulate protein breakdownin erythroc.yte extracts with E and E . 7ooll1 Fig.

pH 9 eluate

from reticulocytes

(concentra se d 30-&d

relative

to the volume of Fraction II, cf. ref. IO) were loaded on a column (0.9 x 60cm) of Ultrogel-ACA-3l, equilibrated with 2OmM Tris-HCl (pH 7.2), 1mMDTTand Img/ml ovalbumin. were collected at /+oC. ATP-dependent I-BSA ( 0-0 ) wasassayedas described under "Methods" with 204 samplesof columnfractions and in the pres&ce of 20~1 erythrocyte lysate. Activities of E ( H ) and E (-1 were assayed with lo-u1 samples of column Q&ions by the quantitative assay for the conjugation of I-ubiquitin, as described under "Methods".

partial.

The presence of residual

is indicated partially

by the observation

Ej in erythrocyte

that protein

restored by the addition

lysates

breakdown is

of E2 alone, while the

decrease of E3 level is indicated by the finding that complete restoration of proteolysis is attained only upon the combined supplementation of E2 and E3. In view of the absence of E2 in erythrooytes,

it was

surprising, at first glance, to find that the conjugation of 125I-ubiquitin with endogenous proteins is not significantly reduced in extracts (Fig. 4, lanes a).

of erythrocytes, as compared to reticulocytes However, when the conjugation of ubiquitin

with exogenous protein

substrates was examined, a striking

difference in the pattern of the conjugates formed was observed. As found previously, proteins which are good substrates for 662

BIOCHEMICAL

Vol. 145, No. 2, 1987

AND BIOPHYSICAL

Retie. III Origin

RESEARCH COMMUNICATIONS

Erythr.

abcdefabcdef

-

Fig. 4. Conjugation of BI-ubiquitin to various proteins in extracts of reticulocytes and er.ythrocgtes. Reaction mixtures contained in a volume of 20u1:50mk'Tris-HCl (pH 2mM ATPI,@ DTT, 0.02 units of ingrganic 7.6), 5fi M&l,, pyrophosphatase, 20 pmol I-ubiquitin (approx. 10 cpm), 2Oug of protein of Fraction II from reticulocytes or erythrocytes, and exogenous protei;is (5ug, each) as follows: lanes a, none; lanes b, lysozyme; lanes c, ribonuclease A with methionine residues oxidized to sulfoxide derivatives; lanes d, a-lactalbumin, reduced and carboxymethylated; lanes e, cytochrome C from S.cerevisae, lanes f, ribonuclease A, oxidized with performic acid. Oxidized derivatives of ribonuclease were prepared as in ref. 12. "Retie" with Fraction II from reticulocytes; VZrythr.", with &action II from erythrocytes. Following incubation at 37'C for 30 min, the samples were electrophoresed on a 12.5%-polyacrylamide-SDS gel.

degradation in reticulocyte

form conjugates with multiple molecules of ubiquitin The formation in reticulocyte extracts (18).

extracts of multiple conjugates of ubiquitin with protein of ribonuclease, substrates such as lysozyme, oxidized derivatives a-lactalbumin ("Retie",

and cytochrome C from yeast is shown in Fig.4

compare lanes b-f to lane a).

By contrast,

in

extracts of erythrocytes only low-molecular-weight conjugates with the same proteins are formed, while the of ubiquitin high-molecular-weight

conjugates are practically 663

absent (Fig.&,

Vol. 145, No. 2, 1987

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

T3ryl2~~~, lanes b-f). This suggests that the ubiquitinconjugating activity observed in erythrocytes is carried out by other enzyme(s) which attach only one, or a few ubiquitin molecules to the protein substrate.

While this work was in progress, Lee et al. (19) reported the separation of multiple forms of ubiquitin-protein ligase from human erythrocytes. The different forms produced mainly the monoubiquitin derivatives of various proteins. E., is required for their action but no requirement for E2 or E3 has been reported (19). It appears likely that the ubiquitin-protein ligase activities which we observed in rabbit erythrocytes (Fig. 4) are similar to those described by Lee et al. (19). Since these ligases obviously do not participate in protein breakdown, their function remains to be established. The conjugation of a single ubiquitin to histones appears to be involved in the modification of histone function, rather than in histone degradation. The ligation of ubiquitin to certain receptors also appears to modify receptor function (2). Thus, ligases such as those observed in erythrocytes may be involved in the modification of the function of various cellular proteins and enzymes. The finding that erythrocytes do not contain a complete ubiquitin ligase system for protein breakdown may help to distinguish between ligases of different functions.

ACKNOWLEDGFMENTS We thank Clara Segal and Judith Hershko for skillful technical. assistance, and Dr. Stuart M. Arfin for helpful comments on the manuscript. This work was supported by Grant AM-25614 from the United States Public Health Service and by a grant from the United StatesIsrael Binational Science Foundation.

1. 2. 3. 4. 5.

Hershko, A. and Ciechanover, A. (1982). Annu. ,Rev. Biochem. a, 335-364. Hershko, A. and Ciechanover, A. (1986). Progr. Nucl. Acid @es. Mol. Biol. 33, 19-56. Muller, M. Dubiel, W., Rathman, J. and Rapoport, S. (1980). Eur. J. Biochem. 109, 405-410. Schweiger, H.G., Rapoport, S. and Schijlzel, E. (1956). Nature 178, 141-142. Hershko, A. Heller, in Protein Turnover Doyle, D.J., eds.).

H., Ganoth, D. and Ciechanover, A. (1978). and Lysosome Function (Segal, H.J. and pp. 149-169, Academic Press, New York. 664

Vol. 145, No. 2, 1987

6. 7. 8. 9. IO. 11. 12. 13. 14. 15. 16. 17. 18. 19.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

McKay, M.J., Daniels, R.S. and Hipkiss, A.R. (1980). Biochem. J. 18% 279-283. - -9 Speiser, S. and Etlinger, J.D. (1982). J. Biol. Chem. 257, 14122-14127. Ciechanover, A., Heller, H., Katz-Etzion, R. and Her&to, A. (1981). Proc. Natl. Acad. Sci. USA. 78, 761-765. Ciechanover, A., Elias, S., Heller, H. and Hershko, A. (1982). J. Biol. Chem257, 2537-2542. Hershko, A., Heller, H., Elias, S. and Ciechanover, A. (1983). J. Biol. Chem. 258, 8206-8214. Pickart, C.M. and Rose, I.A. (1985). J. Biol. Chem. 260, 1573-1581. Hershko, A., Heller, A., Eytan, E. and Reiss, Y. (1986). J. Biol. Chem. 261, 11992-11999. Hershko, A., Lexnsky, E., Ganoth, D. and Heller, H. (1984). Proc. Natl. Acad. Sci. USA. 81, 1619-1623. Ciechanover, A., Elias, S., Heller, H., Ferber, S. and Hershko, A. (1980). J. Biol. Chem. 255, 7525-7528. Haas, A.L. and Wilkinson, K.D. (1985). Prep. Biochem. 2, 49-60. Ciechanover, A., Wolin, S.L., Steitz, J.A. and Lodish, H.F. (1985). Proc. Natl. Acad. Sci. USA. 82, 1341-1345. Hershko, A., Ciechanover, A. and Rose. I.A. (1979). Proc. Natl, Acad. Sci. USA. 76, 310?-3110. Hershko, A., Ciechanover, A. and Rose, I.A. (1979). Proc. Natl. Acad. Sci. USA. 31783-1786. Lee, P.L., Midelfort, C.F., Murakami, K. and Hatcher, V.A. (1986). Biochemistry, 25, 3134-3138.

665