Identical properties of an mRNA-bound protein and a cytosol protein with high affinity for polyadenylate

BIOCHEMICAL

Vol. 80, No. 1, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

January 13,1978

Pages 39-45

IDENTICAL PROPERTIES OF AN mRNA-BOUND PROTEIN AND A CYTOSOL PROTEIN WITH HIGH AFFINITY FOR POLYADENYLATE Gertraud Max-Planck-Institut

MaZUr and Anton

fiir

Biochemie,

Schweiger

8033 Martinsried

bei Miinchen,

Deutschland. Received

July

23,1977

A rat liver soluble protein which binds very tightly to polyadenylate in vitro, and a specific mRNA-bound protein from free polyribosomes, have been isolated by techniques of affinity chromatography. The two protein preparations were compared on the basis of criteria such as stability and specificity of the binding,, molecular weight, which was 78,000 in both cases, and amino acid composition. We conclude that the two proteins are identical or at least highly related species and that the cytosol component may be a precursor of the polyribosome-associated protein.. SUMMARY ::

Polyribosomal two predominant 52,000

protein

species

(P 52) and 78,000

forms a specific at the

mRNA from eukaryotic

and very

indicating

form protein

tight

RNA-binlding identification identifying

factors

must be considered,

polyribosomal Evidence properties

is

There

in a free

in cell

nuclei

weights

however,

with

the poly(A)

are also

reports

soluble

state

carcinoma

in the

of

Such

cells

(7)

We have now extended of cytosol

the

two samples have likely

(5,6).

ascites

as preliminary.

appears

segment

from our laboratory

from a rat

in common and it

P 78

in the pool

and cytoplasm

isolated

that

to

to the poly(A)-associated

work and compared preparations

presented

close

with

Polyribosomal

P 78 and experiments

P 78 in Ehrlich

P 70, both

is associated

(l-3).

complex

in addition

of cytosol cytosol

our pre,vious

that

P 78 exists

of molecular

(P 78) daltons

.3' end of the mRNA (4).

literature

cells

liver

that

P 78 and

homogenate. several

they

structural

are identical

proteins. 0006-291X/78/0801-0039$01.00/0 39

Copyright All rights

0 I978 by Academic Press, Inc. of reproduction in any form reserved

BIOCHEMICAL

Vol. 80, No. 1, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3~1-

MA;;;;A;;~cL~,MEE

53: L3H]-adenosine (40 ci/mmol), [ P -orthophosphate were from New England p Deu E.schland. Nuclear, Dreieich, RNAses A (XII-A), T1 and T2 were products of Sigma, Miinchen. Cellulose-oligo(dT), T3, was obtained from Collaborative Research, Waltham, Mass. .

Male SD rats weighing 200 g were each injected with loo PCi 60 min before sacrifice. The livers were removed I" H I -adenosine and homogenized with 0.02 M Tris-HCl, pH 7.6, 0.05 M NaCl, 5 mM and 0.25 M sucrose (medium A) in a MgC12, 5 xnM 8-mercaptoethanol To prepare free polyribosomes the mitoDounce homogenizer. chondrial supernatant of the homogenate was layered on discontinuous gradients of 0.5 M (1 ml), 1.5 M (1 ml) and 2.0 M (3 ml) sucrose in 0.02 M Tris-HCl, pH 7.6, 0.5 M NaCl and 5 mM MgCl and centrifuged in a Spinco 50 Ti rotor at 50,000 rpm for 5 g. The polyribosome pellets were rinsed twice with medium A. The cytosol fraction was prepared by centrifugation of the mitochondrial supernatant at 50,000 rpm for 3 h (8). In this experiment the animals were previously starved for about 60 h and livers were perfused with a solution of 0.32 M sucrose, 3 mM MgCl in situ. The cytosol was dialysed overnight against the chroktography buffer (0.03 M Tris-HCl, pH 8.4, 1 mM EDTA, 0.1 mM DTE, 0.05 M Nacl and 10% glycerol). Cytosol P 78 was isolated by affinity chromatography on Sepharose-poly(A) essentially as described in our previous report on the purification of P 78 from Ehrlich carcinoma ascites cytoplasm (7). Binding of proteins to the polynucleotide was accomplished in a batch procedure at ice temperature. The gel was then washed successively in a column with 0.2, 0.6, 2.0 M NaCl and 6.0 M urea, each in chromatography buffer, and finally eluted for P 78 with 50% formamide, 0.5 M NaCl in the same buffer. These experiments were performed with a gel prepared from CNBr-activated Sepharose (Pharmacia, Freiburg) and potassium polyadenylate (Boehringer, Mannheim) in our laboratory (8). Commercial preparations of matrix-bound polyadenylate were found to be less suitable. A fraction of polyribosomal P 78 was prepared from free polyribosomes according to Lindberg and Sundquist (9) as modified by Irwin et al. (lo). The material eluted with 5o% formamide, 0.25 M NaCl from a column of cellulose-oligo(dT) was collected and saved for analysis. Poly(A) segments were isolated from the formamide elua e in an experiment in which polyribosomes were labeled with1 3%P] -orthophosphate for 4 h in vivo (11-15). The isolated proteins were analysed by SDS gel electrophoresis (16) on a 7.5% acrylamide slab gel in the E-C Vertical Gel equipment (E-C Apparatus Co., Philadelphia, Pa.). Techniques for the determination of amino acid composition were those that have been described in a previous report (17). The preparation of P 78 from free polyribosomes was incubated with RNAses Tl and T2 at 37O and reprecipitated prior to acid hydrolysis.

RESULTS:

A fraction

as a nearly

pure

described

previously

of cytosol

component (7).

P 78 was prepared

following Pig.

the

procedure

1 and 2A show that

40

from that the

liver we have

protein

in

BIOCHEMICAL

Vol. 80, No. 1, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

%

.MF NoCl - 02

NaCl

NaCl

urea

06

20 r---IT-

60

r

formamlde 50

II II

froctlon

number

Figure IL. Affinity chromatography of cytosol proteins on Sepharose-polyadenylate. Cytosol prepared from 5 rat livers (570 mg protein) was applied to a 7 ml column and fractions of 1.5 ml were collected in a stepwise elution as indicated. o----o , activity: 5 ul of the sample in 1 ml chromatoof protein-bound radioactivity determined (8).

the

formamide

eluate

interacted

during

chromatography:

polyadenylate with

the highest

under that

the the

given

nucleotidek

conditions

could cases.

communication was pre,pared orthophosphate

(8).

for

of all

retained

but

the

the

its

high

with

experiments

labeled

4 h only

with

traces

41

shows

affinity

for

other

was less

in the 0.2

poly-

effective

and 0.6

M

Nacl

assay;

such

in a previous

in which

[3H]-adenosine

of radioactivity

proteins

1 also

certain

have been reported

In control

component

cytosol

Fig.

the binding

obtained

Sepharose-

in the polynucleotide-binding

activity

from livers

with

is thus

of the protein

The material

of binding

it

poly(A)

protein

be observed

active

strongly

of chromatography.

Interaction

washes 'was also profiles

for

formamide-eluted

polyadenylate.

in these

affinity

very

the or

cytosol ["'PI-

were present

BIOCHEMICAL

vol. 80, No. 1, 1978

b

AQ

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

c m ca b m

c d e m

SDS polyacrylamide gel electrophoresis of isolated Figure 2. of cytosol proteins fractionated on proteins. (A), analysis a, material not bound to the gel: Sepharose-polyadenylate: b, c, d, proteins in wash fractions with 0.2, 0.6 M Nacl and e, protein eluted with 50% formamide, 6.0 M urea, respectively: 0.5 M NaCl: m, mixture of molecular weight markers consisting of chymotrypsinogen (25,000). ovalbumin (43,000), bovine serum albumin (68,000) and a-galactosidase (130,000). (B), analysis of polyribosomal proteins fractionated on cellulose-oligo(dT): a, unbound material: b, c, protein eluted with 25% and So% formamide, respectively. (C), comparison of P 78 isolated from free polyribosomes (a) and cytosol (b).

in the sol

formamide

eluate

P 78 was binding A fraction

50% formamide

directly

from rat

not bind

to Sepharose-poly(A)

chromatography.

liver

free

Additional

(Fig.

polyribosomes The material

from the column contained

became bound to the gel via

that

the

cyto-

is bound to mRNA, polyribosomal

on cellulose-oligo(dT).

RNA and one main polypeptide

conclude

to the poly(A).

of P 78 which

P 78, was isolated chromatography

and we therefore

2B).

evidence

Obviously,

42

identical that

eluted

radioactively

the RNA in the under

by affinity with

labeled this

sample since conditions

the polyribosomal

protein it

did of P 78

BIOCHEMICAL

Vol. 80, No. 1, 1978

existed

as a poly(A)-protein

in which

radioactively

characteristic eluted

labeled

size

The yield

of cytosol

fractionation

in the

in the Fig.

phoresis

a

formamide-

P 78. as determined liver.

range of 60-80

migrated

with

shown). in the

on the

Pg per

eluates

after

oligo(dT)

column was

liver.

samples of polyribosomal

as a single

P 78 and cytosol

band when analysed

in a polyacrylamide

slab

component was identical

50% form-

The amount of polyribosomal

polyribosomes

2C shows that

P 78 both

not

in the

25% and 50% formamide

of free

in an experiment

segments of polyadenylate

was 30-50 ug per

P 78 recovered

was obtained

(15) were identified

(diagram

amide eluate,

this

complex

fractions

usually

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

gel.

in both

by SDS electro-

The molecular

weight

of

cases and was calculated

to

be 78,ooo + 2,000. The amino acid Table

1. There

addition large

it

is a great

can be seen that

amino acids

considered

as being

and to what extent proteins column

the

P 78 from

liver,

acidic.

It

dicarboxylic

DISCUSSION: -

used in its

glutamic

protein).

cytosol

almost

Our results

(7),

analyses

and in

relatively the poly-

The ratio

of acidic

therefore

however,

The analysis isolated

in the

(see Table

and corresponding

to cytosol

with

show that

polyribosomal

P 78 and

or identical

properties:

P 78 binds

very

strongly

isolation.

A comparable

43

to the tight

1,

from Ehrlich

identical

similar

be

whether

were present

the

in

(22.8% for

could

is not known,

protein

is given

contain

acid

amino acids

P 78 have a number of

Cytosol

the

preparations

and glutamine.

cell is

between

cytosol

3) of a poly(A)-binding ascites

P 78 proteins

was 1.5 and the proteins

as asparagine

carcinoma

cytosol

both

plus

and 23.0% for

to basic

of the

similarity

amounts of aspartic

ribosomal

(i)

composition

data‘ presented

here.

Sepharose-poly(A)

binding

of protein

to

BIOCHEMICAL

Vol. 80, No. 1, 1978

Table

1.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Amino acid composition of protein P 78. Compositions are expressed in terms of mol per loo mol of recovered amino acids. P 78 isolated

rat liver polyribosomes cytosol

Amino acid

7.4 2.2 4.9 9.4

7.1 2.0 5.6 lo.4

5.0 7.6 13.4 5.9

5.3 7.4

LYS

His Arg Asp Thr Ser Glu Pro

Tyr Phe

the poly(A) authors cytosol

18, 19).

protein

is

7.5 14.4 6.7

6.2

lo.7 8.9 5.1

1.1 4.1 8.3 1.9

1.9 3.2 7.1 3.1

4.0

2.4

The molecular with

of

the molecular

78,000

weight

P 78 closely

The reaction

of cytosol

specific

P 78 from Ehrlich

specificity

weight

by several

of binding

ascites

cells

has been reported

endogeneous protein-polyadenylate

structures

44

(4,

The

that

of

Sepharose-

as has been shown in the

carcinoma

the

(iii)

resembles P 78 with

for

of poly-

by SDS gel electrophoresis.

of cytosol

was to some extent

of cytosol similar

(ii)

identical

composition

P 78 from mRNP. (iv) poly(A)

9.6 5.3

9.0

P 78 as determined

amino acid

6.3 2.2 5.5

segment of hnRNA or mRNA has been reported

(13,

ribosomal

carcinoma ascites cytosol (7)

9.1

5.7 2.3 3.2 7.2 2.7 3.8

Ile Leu

Ehrlich

12.6 6.3

11.2 8.1

GUY Ala Val Met

from

(7).

previously 13, 18).

case

A for

BIOCHEMICAL

Vol. 80, No. 1, 1978

It identical

seems that

the two proteins

or at least

may be the precursor, protein. process

Whether

highly

the

recently

vestigated.

The fact

have been detected

related

or RNA-free free

(20)

for

that in cell

compared

in this

species.

Thus,

form,

protein

of nucleo-cytoplasmic

suggested

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cytosol

for

example,

nuclei

in the

of mRNA, as has been

mRNA-bound P 78, remains significant

P 78

of the mRNA-associated

is involved,

transfer

work are

quantities (5) is in

favour

to be in-

of the

free

of this

P 78 idea.

The authors wish to thank Prof. K. Hannig ACKNOWLEDGEMENTS: for valuable discussion: the technical assistance of Mrs. E. is highly appreciated. Koch andl Mrs. S. Andric

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