Occurrence of a polyubiquitin structure in ubiquitin-protein conjugates

Vol.

128,

No.

May

16,

1985

3, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

OCCURRENCE OF A POLYUBIQUITIN

STRUCTURE IN UBIQUITIN-PROTEIN

Avram Hershko

and Hannah

1079-l

086

CONJUCATES

Heller

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

March

25,

1985

Summary: In the ubiquitin-mediated pathway for the degradation of intracellular proteins, several molecules of ubiquitin are linked to the protein substrate by amide linkages. It was noted that the number of ubiquitinprotein conjugates and their apparent molecular size are higher than expected from the number of amino groups in the protein. When the amino groups of ubiquitin were blocked by reductive methylation, it was efficiently conjugated to lysozyme, but the higher-molecular-weight conjugates were not formed. This suggests that the higher-molecular-weight conjugates with native ubiquitin contain structures in which one molecule of ubiquitin is linked to an amino group of another molecule of ubiquitin. Methylated ubiquitin stimulated protein breakdown at about one half the rate obtained with native ubiquitin, and isolated conjugates of 1251-lysozyme with methylated ubiquitin were broken down by reticulocyte extracts. These findings indicate that the formation of polyubiquitin chains is not obligatory for protein breakdown, though it may accelerate the rate of @ 1985 Academic Press, Inc. this process.

Previous ubiquitin

studies (Ub')

of intracellular are

formed

which

of three

(Ez),

transfer

process;

final

reaction

in Ub-protein

to acid-soluble

molecule

group

of lysine

is

conjugated

conjugates single

are

molecule

'Abbreviations:

of Ub is proteins formed

enzymes:

linked which

in which

of the protein Ub, ubiquitin;

E3, which Proteins

2A by an isopeptide several substrate Me-Ub,

(6).

been sufficiently in which

glycine

to the (7).

E-NH2

multiple

of Ub are bound

(8).

The high-molecular-weight methylated

a

When Ub

degradation,

molecules

reductively

the

to Ub are

enzyme system

linkage for

(2-4);

may catalyze

conjugate

COOH-terminal

substrates

(El),

by a thiol-

conjugated

has not yet

case of a Ub-histone

are

enzyme

transferred

by an ATP-dependent

its

conjugates

of the polypeptide

Ub is

(5).

via

the degradation

a Ub-activating

enzyme,

conjugates

in

Ub-protein

residue

and a third

the

of the polypeptide

event

review).

activated

119 of histone to

1 for

ligation

conjugation signal

glycine

products for

the

to which

of Ub-protein except

single

ref.

COOH-terminal

protein

The structure

that an initial

action

the

characterized,

is (see

ester degraded

indicated

proteins

proteins by the

activates

a Ub-carrier

have

with

to a

ubiquitin. 0006-291X185

1079

All

Copyright 0 1985 rights of reproduction

$1.50

by Academic Press, Inc. m any form reserved.

Vol. 128, No. 3, 1985 Db-protein

conjugates

has been

assumed

substrate

protein

terminal

a-NH2

breakdown

group

group

there

is

preferred

all

Db molecules

but of the

subsequent

was raised

has 6 lysine

residues

tes

the highest

formed,

100,000

in crude

of extremely

groups

possible

are

more than

suggests

that

they

of larger

12 bands apparent

findings

have

been reported

molecular

size.

(6).

highthan Thus, Mr conjuga-

weights

by Rechsteiner

conjugate-degrading the

In the present

may account

for

the

Evidence

are due to the

observation size

molecular

and observed

which

but

the calculated

have

to inhibit

(9),

a-NH2 group

of Ub-lysozyme

of which

causes

protein

formation,

of the protein

of reticulocytes high

by the recent conjugate

"super-high"-molecular-weight-conjugates.

to the protein MATERIALS

which

who used hemin

lysates

for

NH2-

of IJb to the to the

and an a-NH2 group;

yet

Similar

(lO,ll),

we explored

of these which

74,000,

(6).

and co-workers

accumulate of amino

is

for

It

of the

Ub-protein

to E-NH2 groups

question

(6).

a free

essential

conjugation

El + Eg + E3 are used

lysozyme

conjugates

linkages

Another

the number

is

conjugation

occurs.

of Db7-lysozyme

tion,

that

that

from

enzymes

protein

that

of high-molecular-weight suggested

conjugates

degradation to E-NH2 groups

indicates

evidence

expected

above

evidence

no direct

molecular-weight

for

bound

as yet

when purified

are

are

substrate

has been

may precede

substrates

recent

the formation It

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

are (8),

(9).

a-NH2

that

that

and for

conjugates

actually

BIOCHEMICAL

formation

of poly-Db

formation

of

investigastructure is

presented

chains

linked

substrate.

AND METHODS

Dbiquitin was purified from human erythrocytes (5). Crystalline hen eggwhite lysozyme and molecular-weight marker proteins were purchased from sigma. Proteins were radioiodinated by the chloramine-T procedure (8). Preparation of enzyme fractions. Fraction II (a crude enzyme fraction depleted of Ub) was prepared from lysates of rabbit reticulocytes as previously described (12). EI and E2 were purified from fraction II by affinity chromatography on Db-Sepharose and were further purified by gel The source of Eg was the pH 9 eluate of the filtration as described (5). affinity column according to the previously described procedure (5), except that affinity chromatography was performed in the absence of ATP. ES was freed of contaminating Ub by the following procedure: The pH 9 eluate (500 ul, 6 mg of protein) was incubated for 10 min at 37'C with 10 mM dithiothreitol (to cleave residual thiolesters of Ub) and was applied to a l-ml column of DE-52 (Whatman) equilibrated with 3 mM potassium phosphate (pH 7.0) and 1 ml4 dithiothreitol. The column was washed with 10 ml of the above buffer containing 20 mM KC1 (to remove Db), and the enzyme was eluted with a solution consisting of 600 mM KCl, 20 mM TrisHCl (pH 7.2), and 1 mM dithiothreitol. The enzyme was concentrated by centrifuge ultrafiltration with CF-25 Centriflo membrane cones (Amicon), and the buffer was changed by two successive lo-fold dilutions with 20 mM Tris-HCl (pH 7.2) containing 1 mM dithiothreitol, followed by ultrafiltration in the cone. Before use , preparations of E3 were treated with 5 mM iodoacetamide (10 min at 37"C, cf. ref. 5), to inactivate isopeptidases present in the pH 9 eluate. 1080

Vol.

128,

No. 3, 1985

8lOCHEMlCAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Reductive methylation of Ub. Ten milligrams Ub were dissolved in 10 ml of 0.1 M HEPES-NaOH buffer (pH 7.4) containing 6 M urea. Formaldehyde and sodium cyanoborohydride were added to final concentrations of 12 and 20 mM, respectively. The reaction was allowed to proceed at room temperature for 20 hours, following which fresh formaldehyde and cyanoborohydride were added at the above concentrations, and incubation was continued for a further hour. Reagents were removed by dialysis (in Mr = 3500 cutoff dialysis tubing) for 48 hours against 6 changes of water. By this procedure, more than 95% of amino groups of Ub were blocked, as determined by reaction with fluorescamine (13). RESULTS AND DISCUSSION Trying expected

to understand by the number

the possibility

that it

For example, serine

conjugation the

Ub might

with

an amide amino it

cells

which

was conjugated

that

conjugates

enzymatic

with

digests

were

derivative

subjected which that

acid

the label not

residue

in

with

viva

the aid

isolated

shown).

Ub can be conjugated

for

its

to which

free

there

to proteins

linked

cultured

[2,3-3H]glycine,

and it

El + E2 + E3. to prolonged The enzymatic

chromatography.

Thus,

such a

Ub is from

of purified

for

The only

[3H]glycine)

are

Ub

COOH-terminal

aminopeptidase.

(except

with

of Ub labeled

and subjected

analyzer

known

to be consistent

was isolated

and leucine

to amino (data

via

to

the presence

we searched

to the protein acid

(in

appears the aid

with

were

pronase

contained

glycyl-c-NH2-lysine assumption

labeled

to lysozyme

[3H]Gly-Ub-lysozyme

stability

[3~l~ly-~

lysine.

observa-

of lysozyme

to Ub, with

the amino

L3H]glycine. had been

digestion

linked

than to its

at pH 8.8

experiments

linked

Ub is

in vitro

high

In other

residue

was expected to

This

examined

out by the following

conjugates

3 hr).

Since

be attached

hepatoma

was ruled

than

of Ub was linked

in addition

1 M hydroxylamine

linkage.

other

group

linkages,

size

we first

to residues

carboxyl

by ester

with

acid

[3H]glycine.

should

the

This

of higher

of the proteins,

be conjugated

groups.

at 37'C for

hypothetical glycine,

groups

that

residues

to treatment

of 4 M urea,

with

of conjugates

"super-high"-molecular-weight

stable

only

of amino

to amino

tions:

structure

was possible

or threonine

were

the

was

no grounds

in other

than

for

the

amide

linkages. We next conjugates molecule

examined might

of Ub.

by blocking

arise

native

groups

1, the

Ub (Me-Ub)

Ub more than

conjugates

had apparent

Me-Ub only

7 prominent

bands

of Ub to an amino

of such a poly-Ub

with

of conjugates were

chain

weights observed, 1081

those were

group

should with

In the experireductively

of native

Ub.

formed,

and the

above 100,000 the highest

of another

be prevented

methylation.

of 12'1-lysozyme

compared

molecular bands

some of the high-molecular-weight

of Ub by reductive

conjugates

were 12

that

by the conjugation

The formation

the amino

ment shown in Fig. methylated

the possibility

Whereas larger

(lanes of which

with

1, 2) with had an

Vol. 128, No. 3, 1985

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

123456

Figure 1. Conjugation of '251-lysozyme with native and reductively methylated ubiquitin. The complete reaction mixture contained in a final volume of 20 ~1: 50 mM Tris-HCl (pH 7.6), 2 mM ATP, 5 mM MgC12, 3 mM dithiothreitol, 2 ng of '251-lysozyme (approx. 10' cpm), 7.8 nanounits of El, 1.1 nanounits of E2, 1.8 nanounits of Es, and unlabeled Ub or Me-Db as specified. Following incubation at 37°C for 60 min, the samples were separated by SDS-polyacrylamide gel electrophoresis (12.5% in polyacrylamide) and were autoradiographed as described (8). Lanes 1 and 2, with 0.2 I-lg or 3 ng of Db, respectively; lanes 3 and 4, with 0.2 pg or 3 pg of Me-L&, respectively; lane 5, without the addition of Ub or Me-Ub; lane 6, without ATP. Cont., contamination in the preparation of 1251-lysozyme; Lys, lysozyme. The molecular weight markers indicated in the figure are (kDa): 116, S-galactosidase; 97, phosphorylase B; 66, bovine serum albumin; 45, ovalbumin; 29, carbonic anhydrase; 24, trypsinogen; 20, soybean trypsin inhibitor; 14.5, lysozyme.

apparent

molecular

tion

of all

its

derivative

formation

weight

bands

of approximately

required

(lane

of conjugates

(lane

the enzyme preparations gates native

Ub;

weights

the reason migration

may also

account

supplementation apparently that

the

6) and of Ub or

of Ub,

due to slight

there

was some

contamination

migration

of the

gels

observation

due to their that

the

branched

apparent

structure.

molecular

of some Me-Ub-lysozyme conjugates do not correspond to multiples The apparent molecular weights of the conjugates lysozyme.

Me-Ub plus Me-Ub with

lysozyme

are

of

of some conju-

corresponding conjugates of Some conjugates may have known at present.

on polyacrylamide for

The forma-

3, 4).

of ATP (lane

The electrophoretic

from

is not

(lanes

the supplementation

5),

by Ub.

of Me-Ub was different

anomalous This

the Without

5).

50,000

(kilodaltons):

23, 1082

25,

31,

35, 38,

42 and 50.

of of That

Vol.

128,

all

No. 3, 1985

BIOCHEMICAL

the conjugates

contained

the observation obtained

when

shown). amino

The lack

by amide

substrate

shown

for

was only

half is

It caused At least

between

methylated

was indicated

lysozyme in

(data

excess

of native

by

was not

of protein

Ub, strongly

conjugates

the

suggests Ub can be

one of which

is

attached

to

breakdown, that not

of native

rate

of this

should

be noted

a small,

but

part

of this

that

significant

of poly-Ub

chains

was

substrate.

In the experiment

of Me-Ub on the

degradation

of 1251-lysozyme

was examined.

although

obligatory

formation

of the protein

from reticulocytes

on the

its

Ub. for

Me-Ub significantly

effect

This

(at

suggests

protein

breakdown,

saturating that

stimu-

concentrations)

the

formation

although

it

of poly-

may have

an

process. the addition

of ATP alone

stimulation

may be due to residual

Time

~Azyme. Tris-HCl

unlabeled

Ub molecules,

was whether

2, the effect II

protein

effect

linkages

COMMUNICATIONS

of conjugates

conjugates

reductively

the degradation

in Fig.

Ub chains

with

to lysozyme pattern

with

higher-molecular-weight

question

by Fraction lated

was incubated

RESEARCH

protein.

Cur next required

similar

of high-molecular-weight

observed

some of the

formed

BIOPHYSICAL

Me-Ub in addition

an essentially

lz51-Me-Ub

groups,

that the

that

AND

(without

of protein Ub remaining

Ub or MeUb)

breakdown

(Fig.

in Fraction

2). II,

(minuted

Influence of native and methylated ubiquitin on the The reaction mixture contained in a volume of (pH 7.6), 5 mM MgC12, 4 mM ATP, 3 mM dithiothreitol, (approx. 5 x lo4 cpm), and approx. 100 ug of Fraction

breakdown 50 ~1: 1 ug of

of

50 mM 1251-

lysosyme II. Where indicated, Ub or Me-Ub were added at 60 ug/ml. Following incubation at 37°C for the time periods indicated, the release of radioactivity soluble in 5% trichloroacetic acid was determined as described earlier (5). Symbols: A-A, without ATP; A-A, with ATP; Cl-El, with ATP and Me-Ub; C-O, with ATP and Ub.

1083

Vol. 128, No. 3, 1985 which

BIOCHEMICAL

we estimated

protein.

It

by radioimmunoassay

To examine

amino

whether

pure

be intermediates formed

filtration

column. (fraction

was followed molecular

with

native

conjugates

of We-Ub,

breakdown,

Ub-free

range

the

Ub (in

of Lib) and thus

RESEARCH COMMUNICATIONS

1.5 pg Ub per mg of

under

conditions

lack

conjugates

poly-Ub

Me-Uh may breakdown. chains,

isolated

can

with

by a gel-

in Fig. 3. The void free 1251-lysozyme; this

1251-lysozyme-Me-Ub

of 35,000-50,000

protein

of 1251-lysozyme

El + E2 + E3 and were

containing

employed,

some of which

stimulated

which

The preparation is described A) contained mainly aggregated

by a peak weight

that

with

groups

in protein

Me-Ub were volume

conjugates

to free

at around

therefore,

was possible,

Me-Ub may form mixed be linked

AND BIOPHYSICAL

conjugates

(fraction

in

the

B).

Lower-molecularweight conjugates eluted in the following fractions, C and D. The degradation of the protein moiety of isolated 1251-lysozyme-Me-Uh conjugates to acid-soluble

products

vo t

was determined

Hb t

under

OV t

LYS t

conditions

described

earlier

to

ABC0

IS-

5-

Fraction

Number

The reaction mixture of 1251-lysoxyme-Me-IJb conjugates. Figure 3. Isolation contained in a volume of 800 ul: 50 mM Tris-HCl (pll 7.6), 5 mM MgC12, 2 mM ATP, 30 mM Z-mercaptoethanol, 2 ug of yeast inorganic pyrophosphatase (Sigma), 100 pg of 1251-lysozyme (approx. lo* cpm), 60 up of Me-Ub, 310 nanounits of El, of E3 was 44 nanounits of Eg, and 120 nanounits of E3. The preparation purified from residual Ub as described under "Materials and Methods". Following incubation at 37'C for 60 min, the reaction was stopped with 160 ~1 of concentrated formic acid, and the sample was applied to a column (1 x 50 cm) of Sephadex C-150 equilibrated with 5% (w/v) formic acid and 0.1 mg/ml of bovine serum albumin. Fractions of 0.8 ml were collected and radioactivity The fractions indicated was estimated in 20-pl samples of column fractions. (A-D) were collected, dialyzed and lyophilized as described for the preparaVo, void volume; Hb, hemotion of conjugates of Ub (6). Markers (arrows): globin (Mr = 64,000); Ov, ovalbumin (Mr = 45,000); Lys, lysozyme. Inset: SDS-polyacrylamide gel electrophoresis of the pooled fractions, Samples (approx. 10,000 cpm) of fractions A-D were separated on a 12.5% polyacrylamide gel. The numbers on the left-hand side indicate the various in order of increasing molecular size. 1251-lysoayme-Me-Ub conjugates, Cont., contamination in the preparation of 1251-lysozyme. 1084

Vol.

128,

No. 3, 1985

BIOCHEMICAL

Table

1.

AND

BIOPHYSICAL

RESEARCH

ATP-dependent degradation of different '251-lysozyme-Me-Ub conjugates Acid soluble

Fraction

COMMUNICATIONS

classes

of

(%/hr)

-ATP

+ATP

ATP-dependent

ATP-dependent corrected

1.0 6.0 8.8 a.7 3.2

1.2 20.1 14.1 10.2 3.2

0.2 14.1 5.3 1.5 0

0.7 17.1 8.2 3.7 0

A B C D Lysozyme

The reaction mixture contained in a volume of 50 ~1: 50 mM Tris-HCl (pH 7.6), 5 mM MgC12, 3 mM dithiothreitol, 8-15 x lo3 cpm of 1251-lysozyme-Me-Ub conjugate fractions A-D (prepared as described in Fig. 3) or of free Iz51200 ug of protein of the affinity-unadsorbed lysozyme, and approximately fraction of reticulocyte extract (prepared as described in ref. 6). Where indicated, 4 mM ATP was added. Following incubation at 37°C for 60 min, the release of radioactivity soluble in 5% trichloroacetic acid was deterSince free lysozyme is not degraded under mined as described earlier (6). the ATP-dependent degradation was corrected for the these conditions, The contamination of the conjugate preparations by free 1251-lysozyme. contamination of the preparations by free '251-lysoayme was (X of total radioactivity): A, 56.6; B, 12.7; C, 27.8; D, 46.9.

be required with

for

a fraction

enzymes

the breakdown

of proteins

of reticulocyte

extract

and in the presence

considerable mainly (fraction

B).

conjugates

observed

with

conjugates

with

except

that

decreasing of the higher

was

to Me-Ub, conjugates

classes

of Iz51-

molecular degradation

extents

conjugates

of the occurrence clear

is

not the

at present. obligatory

rate.

to an enzyme which

the higher out

may result While that

conjugated

of other

pattern

the high-molecular-weight

is not chains

may increase

ruled

1, there

size.

This

of lz51-

of breakdown

were

of Ub containing

chains.

poly-Ub tes

degradation

decreased (6),

The significance

it

of 1251-lysozyme

Ub, i.e., LJb-conjugating

the higher-molecular-weight

observed

conjugates

to native

of the three

As shown in Table

breakdown containing

the previously

lysozyme-Ub poly-Ub

fraction

The ATP-dependent

lysozyme-Me-Ub resembles

of ATP (6).

ATP-dependent

in the

conjugated devoid

amounts that

the

lower

this

the genes

work

protein

For example,

degrades rates of the

their

of Ub in eukaryotic

protein

of protein chemical

moiety chains.

breakdown modification

reports cells 1085

the

degradation

the binding

in poly-Ub

was in progress,

chains

As shown above, for

of Ub present

from effects

of poly-Ub

in Ub-protein formation

of

to occur,

although

of Ub-protein

may be increased However, observed per

it with

with

cannot

be

Me-Ub

se.

from two laboratories

are present

conjuga-

in multiple

showed adjacent

BIOCHEMICAL

Vol. 128, No. 3, 1985 copies

(14,

15).

This

indicates

in which

the COOH-terminus

terminus

of the

may also

occur

is

cules

are

a single linked

such a fragment is

is

whether

as a polyprotein,

linked

linkage

formed

in Ub at the

to tail"

of the formed

methionine bromide

fragment

synthesized

to

the a-NH2-

to a-NH2 group

poly-Ub,

of Ub

high-molecular-

of 1251-Ub with lysozyme were isolated by preparative and subjected to treatment with cyanogen bromide. cleaves peptide bonds next to methionine residues, and "head

that

nally

Ub is

of one Ub residue To examine

cyanogen

expected

linkage

Ub.

that

in post-translationally

weight conjugates gel electrophoresis Cyanogen bromide there

next

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

size (data

poly-Ub

of free not chains

to E-NH2 residues

NH2-terminus

(COOH-terminus cleavage Ub.

shown),

There

should

If

to NHZ-terminus), produce that

Ub moleit

is

an 1251-labeled

was no significant

indicating

(as opposed

(7).

production

of

in the post-translatio-

to biosynthetic

poly-Ub),

the

of Ub.

ACKNMEDGEMENTS This work was supported by U.S. Public Health Service and by a grant from the United States-Israel Binatfonal Foundation. The skillful technical assistance of Mrs. Clara Segal acknowledged.

Grant AM-25614 Science is

gratefully

REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Hershko, A. and Ciechanover, A. (1982) Annu. Rev. Biochem. 11, 335-364. Ciechanover, A., Heller, H., Katz-Etzion, R. and Hershko, A. (1981) Proc. Natl. Acad. Sci. USA 2, 761-765. Hershko, A., Ciechanover, A. and Rose, I.A. (1981) .I. Biol. Chem. 256, 1525-1528. Haas, A.L., Warms, J.V.B., Hershko, A. and Rose, I.A. (1982) J. Biol. Chem. 257, 2543-2548. Hershko, A., Heller, H., Elias, S. and Ciechanover, A. (1983) .I. Biol. Chem. 258, 8206-8214. H. (1984) Proc. Hershko, A., Leshinsky, E., Ganoth, D. and Heller, Natl. Acad. Sci. USA 1, 1619-1623. Busch, H. and Goldknopf, I.L. (1981) Mol. Cell. Biochem. 40, 173-187. Hershko, A., Ciechanover, A., Heller, H., Haas, A.L. and Rose, I.A. (1980) Proc. Natl. Acad. Sci. USA 11, 1783-1786. Hershko, A., Heller, H., Eytan, E., Kaklij, G. and Rose, I.A. (1984) Proc. Natl. Acad. Sci. USA 8& 7021-7025. Rechsteiner, M., Carlson, N., Chin, D., Hough, R., Rogers, S., Roof, D. and Rote, K. (1984) in Protein Transport and Secretion (ed. Oxender, D.L.) Alan R. Liss, N.Y., pp. 391-402. Hough, R. and Rechsteiner, M. Cell, in press. Ciechanover, A., Hod, Y. and Hershko, A. (1978) Biochem. Biophys. Res. Commun. 1, 1100-1105. Biihlen, P., Stein, S., Dairman, W. and Udenfriend, S. (1973) Arch. Biochem. Biophys. 155, 213-220. ijzkaynak, E., Finley, D. and Varshavsky, A. (1984) Nature 312, 663-666. Dworkin-Rastl, E., Shrutkowski, A. and Dworkin, M.B. (1984) Cells, 321-325.

1086