BIOCHEMICAL

Vol. 87, No. 1, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

March 15, 1979

ACIDIC

PROTEINS

Sdnchez-Madrid, lnstituto

de

FROM

F.,

Saccharomyces

Conde,

Bioquimica

de

C.S.I.C.

Received

281-291

Pages

and

P.,

cerevisiae

Va’zquez,

D.

U.A.M.,

and

Centro

Macromoliculas. Canto

Blanco,

RIBOSOMES

Bal lesta, de

J .P.G.

Biologia

Madrid-34,

Molecular,

Spain

December 28,1978 SUMMARY

Two dimensional gel electrophoresis of ribosomal proteins from Saccharomyces cerevisiae reveals the presence of three spots in the region corresponding to proteins of high acidic character. Washing the ribosomes with 0.4 M NH4Cl and 508 ethanol, followed by chromatography of the extracted proteins on DEAE-cellulose, indicated the presence of two fractions of acidic proteins; (A and Ax), having very similar molecular weights (lZ.OOO-13.000), but phosphorylated to different extents. Fractions A and Ax are immunologically distinct and their immunologic properties differ from acidic proteins found in Escherichia rat 1 iver, and --Artemia sal ina ribosomes. fi, Protein A can be resolved into two bands by electrofocusing, and two dimensional gel electrophoresis. The two components correspond to proteins L44 and L45 according to the standard nomenclature. Proteins Ax seems to correspond to the spot that moves above and to the left of L44 and L45 and is at least three times more phosphoryiated than these two proteins. INTRODUCTION The

presence

to

be

an

isolectric

common

15.000 When one

have these

to

all

point been

(7)

polypeptides.

several types

of

below

pH 5.0

proteins only

among

while

the poor

the homology (10).

in

or

phosphorylation data

proteins with Perhaps

and

from the

of

scarce the

most

the

same

chemical (8,3)

few

properties proteins

weights eukaryotic

in

simple

acidic the

and

acidic

ribosomal

molecular

present

are

strong

Thus,

bacterial

are

differ

with

ribosomes.

in

Although

exist

proteins

reported

acidic

they

copy,

acetylation

rather

of

some

or

12.000

ribosomes

(l-6).

ribosome

in such

two

more

ribosomes from

striking

characteristics

than

as

similarity

proteins

and

unmodified

structural

eukaryotic

having

between

modifications one

seems

seem

studied

Escherichia

(lo),

coli of

to

is these

0006-291X/79/050281-11$01.00/0 281

Copyright All rights

@I 1979

by Academic Press, Inc. in any form reserved.

ofreproduction

Vol. 87, No. 1, 1979

proteins

is

might

the

existance

resemble The

in

E.

some

col

role

i ribosomes in

the

ribosome

role

for

the

however

,

to

identification released

ethanol.

Results

presented

they

in

in

proteins

clarify

the

(15)

and S. or

washing the

S.

AC id ic

be

required

for

are

contradictory

some in

have

ribosomes

of

of the

role required.

We have

cerevisiae

presence

studied

ribosomes of

the

Reports

cerevisiae

three

main

that

(11).

been

ribosomes

functional

molecules

has

factors.

is

myosin

imp1 icated

eukaryotic

their

in

interaction

results, stringent

bacterial

cells

(15).

proteins,

analyzed with

of

a similar

Our

a less

acidic

detail

ribosome

suggest (3).

great

the

their acidic

ammonium-

phosphorylated

proteins

report. AND METHODS

ribosome cerevisiae in low were

The-cells described

are

than

purification

this

to

in

as

there

supernatant

MATERIALS

Cells

seem

Saccharomyces

showing

such

proteins

they

acidic

by

regions

although

proteins

and

proteins

these

and

several

that

for

In order

of

synthesis,

acidic

indicate

requirements

alanine-rich

Specifically,

with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

proteins

(12)

protein

(13,14).

reports

of

contractil

functional

activity

are

BIOCHEMICAL

Y166 was phosphate co1 I&ted,

grown medium broken.anb

to

(16)

late exponential phase when 32~ labeled cells the ribosomes prepared

in YEPD medium were reauired. as previously

(15). proteins

The acidic proteins were extracted by washing the ribosomes with 0.4 M [email protected] in the presence of 50% ethanol at 0°C (according to Hamel et al. (17)) with the modif ications reported (15). The proteins extracted (SPO.4) were applied to DEAE-cellulose column and eluted essentially in the same condit-ions described for Artemia sal ina proteins (3) but using a steeper gradient (10 mM to 1.5 M)ofium acetate. Proteins were labeled with 125lodine in the presence of chloramine T, when required (18). Electrophores

is

Two

dimensional gel electrophoresis was carried out according to standard system of Kaltschmidt and Wittmann (19) with some modifications (20). When SDS was present in the second dimension, the conditions of Martini and Gould (21) were used. Electrophocusing was done using LKB ampholines between pH 3 and pH 5 in tubes of 10 cm x 0.5 cm following pub1 ished conditions (9).

282

the

BIOCHEMICAL

Vol. 87, No. 1, 1979

When required, spots (22). The absorption estimated.

acid were

Immunological

were at

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

eluted

605 nm and

with 67% acetic of the sample

from the gel plates the radioactivity

techniques

Antisera were rised in rabbit by intramuscular injection of the proteins in complete adjuvant followed by two subcutaneous booster injections in incomplete adjuvant every 15 days. Sera were processed as described (15). lmmunodifusion tests were carried out on 1% agar plates, in 10 mM Tris-HCI, 150 mM NaCl pH 7.4.

125

Radioimmunoassay was carried out using l-labeled protein Ax and anti-SPO.4 bound to CNBr activated Sepharose 4B. About 50000 cpm of labeled Ax (specific activity 18500 cpm/pmol) were added to 40 pl of antiSPO.b-Sepharose (1 mg/ml) and after incubation for 1 h at room temperature and 1 h at 0°C the samples were centrifuged at 5000 rpm for 10 min. The pellet was washed twice with water and finally resuspended in 100 ~1 of water. The bound radiactivity was measured in 50~1 of the final suspension. Competition was done incubating in the presence of increasing concentration of unlabeled proteins.

RESULTS

Two of

S.

dimensional

cerevisiae

character of

shows

moving

E.

coli

have

in

the

L44/45

and

~35/36

detect

the

presence

that

does

and

d).

the

acidic

not When

times

washing

and the

higher

acidic them

the

with

0.4

of

(23)

and

that

region

two

Planta spots

in

second

the

region

dimension

gel

is

resolved

to

nomenclature. in

dimension

carried

into

two

of

spots

in

ri’bosomes

strong

acidic

proteins

Zinker

and

L7 and

L12

Warner

(24)

that

they

named

However,

we

frecuently

L44/45

and

lA,

spots

(Fig.

out

80s

the

with

a fourth a,

presence

b,

of

apparently

c,

SDS,

similar

1B).

the

in

the

presence

specific at

spot can

M NH4Cl

of

activity

absorption in

proteins

spots

and

spots

obtained

to

The

in

(Fig.

radioactive

radioactivity

three

several

from

similar

respective

their

proteins

a position

in

are

ribosomes

to

of

second

weights

d are

gels

of

presence

enter

of

presence

Krujswijk

proteins

molecular In

the

of

the

electrophoresis the

ribosomes.

reported

three

gel

c be and

605

calculated nm

(Table

in

the

a,

from

eluted

b,

the

c,

ratio

spots,

is

at

from

the

ribosomes

When

proteins

and of least

1).

selectively 50%

32,,3- 4 , spots

removed ethanol

283

(15).

thus

by

Vol. 87, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a

284

BIOCHEMICAL

Vol. 87, No. 1, 1979

Table

1.

Distribution

of

radioactivity

dimensional

among

gel

32 P labeled

spot

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

acidic

proteins

in

Mixture

proteins CPM/A605

A605

‘251-

of

labeled CPM

two

electrophoresis

Ax

and

labeled

un-

A

wm

a

496

0.024

20.6 x lo3

291

b

663

0.028

23.6

866

0.018

60.6 x 103

0.024

24.2

1092

C

d

581

extracted

from

ribosomes

DEAE-cellulose

as

phosphorylated

proteins,

ratio

described

32 P/protein

heavily

in

labeled By

than

of

C marker

or

components

several

fraction

whereas b and Ax,

is seem

proteins in

Ax

fraction

different

spot

was

shown).

32P

and

Methods,

Ax,

were

were

chromatographed two

peaks

obtained

indicated

two

is

of

made made

to

that

Ax

in

of

(Fig.

the

origin

in

very

dimensional

of

(21%)

2).

is

The

more

with

the

second

both

are

to made

molecular

spot

to

the

c.

Both d,

dimension

Ax

close

in

the

a of

weight

one (about

indicated

corresponding

spot of

A and

electrophoresis

moving

formation of

similar

polypeptide

spots

of

a position that

gel

one two

a mixture

suggests

or

only

the

of

This

contaminated

in

939

with

detected

identical

However

contribute

remain

lo3

electrophoresis

of

A is

slightly to

clearly

2666

A.

(not

da1 tons).

that

fractions

a single

cytochrome

Materials A and

103

x

vivo

fractions

gel

SDS,

--in

in

the

polyacrylamide

presence

13.000

labeled

x

position fractions,

thus

part in

to

spot

of

a and

c,

A and of

variable

these amounts

electrophoresis.

Figure 1. Two dimensional gel electrophoresis of 805 ribosomal from S. cerevisiae. A) Second dimension in standard conditions. dimension in the presence of sodium dodecylfulphate.

285

proteins B) Second

Vol. 87, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

‘=Z /’

A

9 IOmM-,

I’

4.0

10

20

30

FRACTION

0 g a.

100

n?

Figure 2. Chromatography on DEAE-cellulose of SPO.4 fraction from 80s r i bosomes . Proteins from the ammonium-ethanol treatment (SPO.4) were applied to a column of 2.5 cm x 20 cm and eluted as indicated in Methods. Radioactivity and protein concentrations were tested in the fractions.

These Ax

and

results

were

unlabeled

proteins

electrophoresis. mainly

to The

sample isolectric three

As spot

main

whose occasionally to

their

from

indicated

of

80s in

fraction

Ax

polyacrylamide

gels

point

close

bands

detected

electrophocused

by

resolving

a mixture

ribosomes

Table

by

two

l-labeled

dimensional

radioactivity

1,

125

of

is

associated

c.

purity in

confirmed

in

intensities

the vary

detected dephosphorylated

to

was between

pH 4.0 when

and the

conditions.

with

the below

electrophocusing pH

corresponds

larger

products.

286

SP

of

the

ones,

the

3). most

proteins

the

the

3 (Fig. to

acidic In

preparation the

by

pH 5 and

total

same

just

confirmed

0.4

ribosomes, probably

has

acidic

(SPo samples

Ax

4)

an of

the

are

faint, are corresponding

bands

BIOCHEMICAL

Vol. 87, No. 1, 1979

Figure Fraction

3.

Electrophocusing SPOe4.

Using possible when is

antisera to

in

with

appears

same

conclusion

and fraction

We have cross

reacts

the

fractions

l-labeled

the

a double can

Ax.

Ax

be

As

1,

acidic give

(Fig. from

as

gels.

total

A and

immunoprecipitation

fact

125

polyacrylamide

against

distinguishable

The SPOs4

that

by

clearly

latter

rised

show

tested

in

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

4).

Fraction

proteins

different The

Ax.

(SPU.,,)

it

precipitation

precipitation

precipitation

2,

band

of

A

was bands

of

band

and

the

Ax

band. drawn

shown

from in

a radioimnunoassay 5 fraction

Fig.

using Ax

does

not

anticompete

A. previously with

antisera

shown rised

that

neither against

(15).

287

of proteins

the

two

fractions

L7 and

L12

A and from

E.

Ax coli

BIOCHEMICAL

Vol. 87, No. 1, 1979

Figure 4 and

4. Double 6, fraction

immunodifusion SPD.4. Well

3,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

test. Central fraction Ax.

L

10’2

well, Well

anti-SP 5 fracti%4A.We’1s

I

10”

I

100

[email protected] Df PROTEIN UNLIBELED

288

10’ -O-Q-O-CC.-

A A,

29

Vol. 87, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DISCUSSION Our three a,

results

main must

L45

and

part

of

of both to

proteins

strong

henceforth

we will

60s

subunit

are

similar

in

different

two

degrees and

Al

and

A2,

are

chemical

fore

possible

that,

if

the

This eL12, the

extent

previously

L44/45

pro&in,

in

yeast,

and

Warner

Protein

Ax

and

variants

of

at Ax

is

in

least

clearly

to

although

it

by

forms fraction might

three

of

Ax,

is

there-

the

site

factor. two

have

to

two

that

process,

not

from

of

It

proteins, been

a single

has

correspond

eL7

shown

to

and be

polypeptide

been spot

(9).

reported PI’ detected s

by

(24).

weight

very

specific times

L45

data

polypeptide.

where

be

represent

suggest

differentiating

and

they

L44/45,

the

that

must

composition

is

autoradiography

high

acid

modifying

L44

find

However,

the

form

dissociation we

that

is

salina,

unphosphorylated c or

its

A.

of

to

and

32 P incorporated

protein.

a single

modification

L44

phosphorylation

amino

equivalent

a molecular

therefore

phosphorylated However,

has

the

b and

They

upon

unlikely

same

probably

spot

only

of

seems

on

as

(24),

ratio

of

spots

nomenclature.

reports

the

the

8)

this

other

cerevisiae

them,

released

extension

of

of

S.

previously

partially

it

of

Two

using

from

similar and

third

the

different

ribosome

named

with

ref.

characteristics

The

Zinker

the is

phosphorylated

be

phosphorylation

of

situation

with

to

Hence

in

proteins

not

them

modification

both

and

to

and

spots,

80s

proteins

agreement

(reported

proteins,

seem

molecules. of

Osawa

refer

phosphorylated the

the

properties.

to

In

of

the

in

acidic

and

(25).

intensity

yeast

presence

respectively

proteins

ltoh

of

ribosomes

the

the

correspond

the

the

show

close

activity more

distinguishable

than from

or

identical

indicated the

other

L44/45

in

that acidic its

to

proteins

it

is

proteins.

antigenic

125 i-labeled Figure 5. Radioimmunoassay. fraction Ax and anti-SPo 4, to Sepharose 4B, were allowed to react in the presence of increasing concentrations of unlabeled fraction A (0) or fraction Ax (B). Results obtained with fraction A were corrected for the 21% contamination of

289

bound

Ax.

Vol.

87,

No.

determinants, with

BIOCHEMICAL

1, 1979

and

characteristics

proteins

and phase.

proteins

The indicate

results

L44/45

L12

that

ti

L41 and

in

in

in

It

present

in

having

different

the

to

namely

A.

sal

ina

to

be

related

L45

and

Ax

results

from

ccl

with

Is

the

a protein to

the

change

indicate

in

in

that

stationary

changing

species having

proteins

L7/12 report).

one

or

Ax

growth ratio

“top

All

of

several

or

whether

polypeptide-two

and

band” them

these

P2

of

in are

A.

ways

system

(8),

(9),

and

ina

to

to

nine

in

proteins of

found

rat

are

ribosomes ccl

detail

1 iver

susceptible

eukaryotic

proteins

rat

sal

up

types

some

immunigenic

in

modified

several some

in or

derivatives, all

In

studied chemical

Pl

and

whether

proteins.

one

iminary

proteins,

known

L44,

eukaryotic

acidic

ribosomes

prel

agreement

four

(4),

yet

COMMUNICATIONS

(26).

cell,

acidic

duplicated

i

(this

not

In

suggested

proteins

ribosomes in

col

cells

been

three

in be

the

yeast

is

all

exist

1s

seem

to

in

bacterial

Is. Nothing

acidic on

L45

of

has

Our

originating

(5).

have

the

from

HeLa

Ax

of

E.

types

phosphorylation 1 iver

in

available

exist

and

Ax

RESEARCH

(9).

would

data

differences

protein.

L44/45

over

L7 and

be a different

preparations.

predominate These

BIOPHYSICAL

to

amounts

ribosomal

L44

L40

to

relative

different

to

similar

equivalent The

ccl

appears

AND

is

known

proteins. these

this

is

In

acidic so,

function

that

protein

species

about our

proteins

the

experience than

proteins

might

is

compatible

with

a more

of

eukaryotic bacterial

these

in

significance

sophisticated

have the

such

a diversity

ribosomes ribosomes

for

a modulator existance cell

are

less

functioning

effect of

of

a larger

on

ribosomal

dependent (15).

the

ribosome

number

of

If

acidic

machinery.

AC KNOWLEDG EHENTS This Biologia (Instituto Laboratories

work was supported by an institutional Molecular from ComisiBn Administradora National de PrevisiBn) and persanal and Lilly Indiana of Spain.

grant to the Centro de de1 Descuento Complementario grants from Essex

Vol. 87, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1. 2.

M?iller, 25,5-12. Stgffler,

71,

W.,

Groene, G.,

Wool,

A.,

Terhorst,

I.G.,

Lin,

C. A.

and

and Rak,

Amons, K.H.

R.

(1372)

(1974)

Eur.J.Biochem. Proc.Nat.Acad.Sci.

4723-4726.

3. Mb'ller, 4.

5. 6. 7. 8. 3. 10. 11.

W., Slobin, L.I., Amons, R. and Richter, D. (1375) Proc.Nat.Acad. Sci. 72, 4744-4748. Horak, I. and Schiffmann, D. (1377) Eur.J.Biochem. 79, 375-380. Reyes, R., V6zquez, D. and Ballesta, J.P.G. (1377) Eur.J.Biochem. 73, 25-31. Leader, D.P., Coia, A.A. and Falmy, L.H. (1978) Biochem.Biophys.Res. Commun. 83, 50-58. Terhorst, C., Mgller, W., Laursen, R. and Wittmann-Liebold, B. (1973) Eur.J.Biochem. 34, 138-152. Tsurugi, K., Collatz, E., Todokoro, K., Ulbrich, N., Lightfoot, H.N. and Wool, 1-G. (1378) J.Biol.Chem. 253, 946-955. Van Agthoven, A., Maassen, J.A. and Mb'ller, W. (1977) Biochem.Biophys.Res. Cotrunun. 77, 989-998. Amons, R., van Agthoven, A., Pluijms, W., Mb'ller, W., Higo, K., Itoh, T. and Osawa, S. (1977) FEBS Letters 81, 308-310. Amons, R., van Agthoven, A., Pluijms, W. and Mb'ller, W. (1378)FEBS Letters

86, 282-284. 12. 13. 14. 15. 16. 17.

18. 19. 20. 21. 22. 23. 24. 25.

Mb'ller, W. (1374) In "Ribosomes" (M.Nomura, A.TissiEres and P.Lengyel, eds.), pp. 711-731. Cold Spring Harbor Laboratory, New York. Glick, B.R. (1377) FEBS Letters 73, l-5. Koteliansky, V.E., Domogatsky, S.P., Gudkov, A.T. and Spirin, A.S. (1377) FEBS Letters 73, 6-11. Ssnchez-Madrid, F., Reyes, R., Conde, P., VLzquez, D. and Ballesta, J.P.G. (1973) Eur.J.Biochem. (submitted). Rubin, G.M. (1873) J.Biol.Chem. 248, 3860-3875. Hamel, E., Koka, M. and Nakamoto, T. (1372) J.Biol.Chem. 247, 805-814. Greenwood, F.C., Hunter, W.M. and Glover, J.S. (1363) Bi0chem.J. 83, 114-123. Kaltschmidt, E. and Wittmann, H.G. (1370) Anal.Biochem. 36, 401-412. Howard, F.A. and Traut, R.R. (1374) Methods Enzymol. 30, 526-533. Martin, O.H. and Gould, H.J. (1571) J.Mol.Biol. 62, 403-405. Hardy,S.J.S. (1375) Mol.Gen.Genetics 140, 253-274. Kruiswijk, T. and Planta, R.J. (1375) FEBS Letters 58, 102-105. Zinker , S. and Warner, J.R. (1376) J.Biol.Chem. 251, 1739-1807. Kruiswijk, T., Planta, R.J. and Mager, W.H. (1378) Eur.J.Biochem. 83, 245-

252. 26.

Ramagopal,

S.

and

Subramanian,

A.R.

(1374)

291

Proc.Nat.Acad.Sci.

71,

2136-2140.

Acidic proteins from Saccharomyces cerevisiae ribosomes.

BIOCHEMICAL Vol. 87, No. 1, 1979 AND BIOPHYSICAL RESEARCH COMMUNICATIONS March 15, 1979 ACIDIC PROTEINS Sdnchez-Madrid, lnstituto de FROM F.,...
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