Characterization of two different messenger ribonucleoprotein particles isolated from A postpolysomal fraction of the rabbit reticulocyte lysate

Vol. 99, No. 4,1981 April

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages 1108-l

30, 1981

CHARACTERIZATION ISOLATED FROM

OF TWO DIFFERENT MESSENGER RIBONUCLEOPROTEIN PARTICLES A POSTPOLYSOMAL FRACTION OF THE RABBIT RETI CULOCYTE LYSATE

Wolf-JUrgen lnstitut

Received

116

fiir

Heidemarie

Euhl,

Biologie Ill D-7800 Freiburg,

February

der

Ernst

and

Kurt

Hilse

Universitat Freiburg, Schanzlestr. Federal Republic of Germany

1

2,1981

Using two different approaches two types of 25-305 messenger ribonucleoprotein particles have been isolated from a rabbit reticulocyte postpolysomal fraction whose mRNA is included in 50s complexes composed of the 40s ribosomal subunit, initiation factors, and Met-tRNAf. The two types of particles differ .in their protein composition; one has a pattern almost identical with the protein pattern of the free cytoplasmic messenger ribonucleoprotein. At least the with the distinct protein composition is translatable in vitro. Posparticle sible relationships between the mRNA-containing 50s complexes and the different messenger ribonucleoprotein particles will be discussed. The is

initiation

generally

elF-3,

GTP

(4-6).

tiation first

joining

of

absence

It

(7).

of

free

60s

It

is

contains in

the

a translatable In

types

at

thought

this of

Abbreviations:

50s

(200 has

been

40s

ribosomal

subunit,

particles

initiation

and

the

The

the

complex

which

the

sediments

formation

hydrolysis to

factor

ternary

complex in

lysate

of give

the

the

mRNA-containing

of

an

final

80s

ini-

preinitiation

complex

which

en-

elf-z-bound

com-

decomposes

in

the

(8).

000

x g fraction)

isolated

the

The

(1,2),

subunit

that

reticulocyte

(l-8):

results

(4,6).

subunits

represent

form

mRNA

rabbit

a new

intermediate

to

communication 25-30s

possible

the

form

ribosomal

a labile

fraction

sedimenting

(3)

of 485

60s

ribosomal

A postpolysomal plex

GTP

at

the is

into

steps subunit

binding

sediments

in

several ribosomal

subsequent

converts

translation

and

which

complex

plex

involve

Met-tRNAf

The

the

mRNA

a 40s

elF-2,

complex allows

to with

of

43s

larged

globin

believed

associated

consisting at

of

from

mRNA-containing elF-2

which rabbit

is

preinitiation and

enriched

reticulocyte

elF-3

complex,

as

well

in

a com-

lysates

as

(9).

since

a-globin

it

mRNA

(10). we

describe

which

both

two contain

procedures mRNA

for but

differ

the

isolation in

their

of

two

protein

elF, eukaryotic initiation factor (nomenclature as devised by the International Conference of Protein Synthesis, Bethesda, Md., USA, 1976 (23)); Met-tRNAf, initiator methionyl transfer RNA; GuoPP(CH2)P, guanosi ne 5 ‘-(B,y-methylene)triphosphate; DTE, dithioerythritol; SDS, sodium dodecyl sulphate; (c)mRNP, (cytoplasmic) messenger ribonucleoprotein; M,, apparent molecular weight of proteins in SDS.

0006-291X/81/081108-09$01.00/0 Copyright @I 1981 b.v Academic Press. inc. All righfs of reproduction in any form reserved.

1108

BIOCHEMICAL

Vol. 99, No. 4,1981

patterns. of

the

They 200

000

are

assumed

to

be

AND

BIOPHYSICAL

derived

from

the

RESEARCH

mRNA-containing

COMMUNICATIONS

50s

complexes

x g fraction. MATERIALS

AND METHODS

Materials. Crude initiation factors and the Ehrlich Ascites cell-free exThe isolation of the postpolysomal tract were prepared as described in (10). 200 000 x g fraction was carried out as detailed in (9). Heparin-sepharose 66 was kindly donated by H.O. Voorma (Utrecht, The Netherlands). Purified elF-2 and elF-3 from rabbit reticulocytes were generous gifts of T. Staehelin (Basle, Switzerland). Rabbits were made anemic by five Preparation of rabbit reticulocyte lysate. daily subcutaneous injections of 2% (w/v) acetylphenylhydrazine (0.45 ml per kg body weight). After a rest period of one day each rabbit was anesthesized with 0.35 ml per kg body weight of a pentobarbital-heparin solution (110 mg pentobarbital and 600 units of heparin per ml) and exsanguinated by heart puncture. The blood was diluted with 2 volumes of ice cold physiological saline and centrifuged at 3 500 x g for 10 min. The packed cells were resuspended, washed three times, and further treated according to Schimke et al. (11). Incubation of the 200 000 x g fraction with GuoPP(CH2)P. Seven volumes of suspended in TKMD buffer (20 mM Tristhe 200 UOO x g fraction (10 A260 units), HCI, pH 7.0, 30 mM KCI, 1.5 mM MgCl2, 0.25 mM DTE), were incubated at 340C for 35 min with 3 volumes of a 'master mix' (20 mM Tris-HCI, pH 7.0, 40 mM KCI, 3 mM MgCI2, 0.5 mM ATP, 15 mM creatine phosphate, 25 IU/ml creatine phosphokinase, 1 mM GuoPP(CH2)P). The incubated reaction mixture was then diluted with 6 volumes of ice cold TM buffer (20 mM Tris-HCI, pH 7.0, 1 mM MgCl2) to a final volume of 400 ~1. Preparative sucrose gradients. The preparation of the gradient for the analysis of the 200 000 x g fraction which was incubated with GuoPP(CH2)P was performed according to Peterson et al. (2). The sample (400 ~1) was layered over a 12-ml linear sucrose gradient (15-305 (w/v)) in TKMG buffer (20 mM Tris-HCI pH 7.0, 10 mM KCI, 1.5 mM MgCl2, 0.1 mM GuoPP(CH2)P). Centrifugation was carrfed out at 40 000 rev./min for 4.5 hr in an SW41 rotor (Beckman) at 4OC. Sucrose density-gradient analyses of untreated 200 000 x g fractions were carried out using the method of Jacobs-Lorena and Baglioni (12). The fractions were suspended in STMDE buffer (250 mM sucrose, 10 mM Tris-HCI pH 7.5, 5 mM Mg-acetate, 1 mM DTE, 0.25 mM EDTA) to give a final concentratfon of 35 Ai units/ml, and 1.5 ml (complemented with 7.5 ml of paraffin oil) were applied onto a 28-ml linear sucrose gradient (20-40s (w/v)) in TKM buffer (10 mM TrisHCI, pH 7.5, 20 mM KCI, 1.5 mM MgCl2). Centrifugation was carried out at 25 000 rev./min for 30 hr in an SW27 rotor (Beckman) at 4oC. The 12-ml gradients were fractionated into sixty 0.21-ml fractions, and the 28-ml gradients into fourty-seven 0.63-ml fractions. Absorbance at 260 nm was monitored simultaneously followino the method of Schreier et al. (13). Characterization of'gradient-derived fractions. Samples of selected fractions were assaved for stimulation of protein svnthesis in the Ehrlich Ascites system as described in (10). mRNA was detected by [51-3H]poly(U) hybridization following the method of Safer et al. (6) except that [5'-3Hlpoly(U) (296 pCi/ pmol of phosphate) equivalent to 68 pmol of uridine (approxjmately 13 000 cpm) was added to each assay (110 ~1). The peak fractions were subsequently pooled and prepared for SDS/polyacrylamide gel electrophoresis: The material was dialyzed against 7.5% (v/v) propionic acid containing 7 mM DTE, lyophylized, dissolved in sample buffer (Laemmli (14)) and subsequently heated to 100°C for 2 min. Electrophoresis was carried out in slab gels using the buffer system of Laemmli (14). The postelectrophoretic procedures were the same as described by Mumby and Traugh (15). Particular peak fractions were analysed for the presence of initiation factors by means of Heparin-sepharose chromatography whose conditions were adopted from van der Mast et al. (16).

1109

8lOCHEMlCAL

Vol. 99, No. 4,198l

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

fraction in Fiq.l: Sucrose density-gradient analysis of the 200 000 x presence of GuoPP(CH2)P. 10 A260 units were analysed (-e,. Portions selected fractions were assayed for protein synthesis (40 ul; background corporation 450 cpm; D.---O) and for [5’-3H]poly(U) hybridization (70 ul; the sedimentation background i ncorporat ion 40 cpm; o.-0 1. To estimate ues of the different constituents of the 200 000 x g fraction, 4.5 Ap60 of polyribosomes were centrifuged in a parallel gradient to indicate the sitions of ribosomal subunits and SOS ribosomes (-).

the of invalunits po-

KESULTS In

a previous

lysate

of

‘top

the

fraction’

particles

report 200

000

and which

50s

were

we

described

the

x g fraction particles, found

to

isolation

which both

be

from

contains active

inactive

three in

rabbit main

protein

4OS:6OS

reticulocyte constituents:

synthesis,

ribosomal

a and

subunit

80s

couples

(9,10,17). It ation

was

suggested

complexes

the

200

000

GTP

analogue

that

the

50s

particles

In

order

to

enhance

x g fraction

was

incubated

in

GuoPP(CH2)P

prior

to

sucrose

(10).

the

1110

represent their the

mRNA-containing

stabilization

and

presence

of

the

gradient

centrifugation

preinitiaccumulation,

nonhydrolyzable (Fig.1).

BIOCHEMICAL

Vol. 99, No. 4,198l

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

-2

TOP

Fraction Fig.2: InngPd selected ground

Number

density-gradient analysis of the 200 000 x g fraction; protime. 50 A~60 (units were analysed C---j. Portions of were assayed for [5’-%)poly(UJ hybridization (50 ~1; backFor the estimation of sedimentation val160 cpm; o -. -0). units cf polyribosomes were analysed in a parallel gradient

Sucrose centrifuqation fractions incorporation

ues 45 A260 C---J.

Besides are

active

now

ciated uents

arise

latter the most

able with

being gradient of

material to

stimulate

the in

50s the

fractions.

the

top

protein

40s in in

and

the

gradient

in

the

two

25-305

synthesis. was

Hybridizable

80s

while

Additionally,

protein Fig.1

of

synthesis,

particles.

active shown

its

at

almost

of

The

localization

material

1111

by

no

previously

regions

determined

particles

the

r57-3H]polyW was detected

occur activity

is

unobserved absorbance of

prof

poly(A)-mRNA

the

assoconstit-

hybridization in

which

ZO-3OS,

i le,

the in to

40-60s

6IOCHEMICAL

ksl. 99, No. 4,1981

AND

BIOPHYSICAL

assessment

of the mFZNA contained 200 000 x g fraction.

conditions for the gradient centrifugation of the 200 000 x g fraction with respect to the 50s complexes

COMMUNICATJONS

1

Table

Quantitative

RESEARCH

in

constituents

of

[5’-3Hjpoly(U) hybridizable

region of the gradient

the

mRNAX [pm'1 -.-

20- 30s (Fr.37-49)

stabi I iz (Fig.1

127

15.1

120

14.2

n9

50s (Fr.21-30)

--

20- 30s (Fr.16-27)

destabilizing (Fig.21

(Fr.

'

and

1 260

50s 3-10)

149

625

74

1 pmol of poly(A) hybridized 8 400 cpm of 1 5'-3H ]poly(U) poiy(A) tract length of 43 nucleotides (6). Furthermore, that the poly(A) tracts of globin mRNAs or mRNPs hybridize with the same efficiency as free poly(A).

80s

regions,

whereas

the

‘top

fraction’

seems

to

assuming an average we have assumed ~5'-3Hlpoly(U)

be

free

of

any

poly(A)-

mRNA . I n order fraction,

(Fig.2).

located

at

serve

two

poly(U)

the

top

types

of

in

lents

of With the

mRNA

of

of

the

fol

lowing

et

al

.

(121.

ty

to

Table

in

gradient

pro prote

mRNA is

is

000

30s.

are we

From

associated

the

to

ob-

[51-3H3-

with

adjacent

x g

centrifumaxima

Furthermore, and

found

200

gradient

absorbance

gradient. 20s

the

the

the

40s

20-30s peak

gradients

Fig.

1 and in

the Fig.2

This

method

t ein

of

fractions

was

summed

which and

correspond

expressed

to

as

equiva-

1).

mRNP

standard The

the

that

of

sucrose and

the

material both

shown

cytoplasmic the

obvious

complexes

the

between

hybridization

of

(Fig.3B,F,G).

free

of

pelleted of

SDS/polyacrylamide

shown

the

of

50s

le.

regions

of

time now

region

hybridizable

(pmol; aid

patterns

becomes

amount

50s

the

405

ni ng

sedimenting

it

profi

gradient

25-30s) tein

or

the are

the

particles

ler

mRNA-contai

particles

[5’-3Hlpoly(U)

20-30s

the prolonged

80s and

absorbance

The

i lari

The

A smal

the

the

I ize

hybridization

material.

of

destabi

we considerably

gation

of

to

elect

rophoresis

two

partis

cles

were

comparison

CcmRNP) of

the

the other

1112

the

includes

furthermore

from

20s three

25-30s

20-30s

with

preparation of

of

of

the

compared

isolated

mRNP

composition

i n patterns

gel

respect

the

rabbit

reticulocyte by

particles

region to

described particle

particle (20s;

their

pro-

proteins lysate

Jacobs-Lorena

(Fig.3F)

shows in

question

no

sim-

(Fig.

BIOCHEMICAL

Vol. 99, No. 4,1981

AND BIOPHYSICAL

RESEARCH

COMMUNICATIONS

150-160 120-130 ‘iw;: 64 64 -68 55

58

45

- 49

40 35

-42 -38

64 55

-68 -58

45 40 35

- 49 - 42 - 38

21

-23

21 - 23

- 6CJ .. 58

45

- 49

40 35

- 42 - 38

21 - 23

I

A

6

A,E,I: B -

cl: B: C,D:

F - H: F: G: H: K: L,M:

3B,G,K). and

C

Their

30 000,

patterns where

polypeptides the

comparison

and

70

cmRNP

(track

pattern

of

with

the

particles

in

tides

with

units

of

the elF-2

identified

25-30s

as

in

000

I

I

H

the

question

38

do

not

same

the

elF-3

of

set

= 72

K

L

and

also

most have

occur. the

chromatography,

50s where

bound

of

76

range

three

proteins. 6)

at

M

elF-2,

initiation

factors

of

gradient bound

contains

a Mr=72

the

since

Both

000

the

three

sub-

could

be

the resin

-

three

polypep-

the

1 with to

these

molecular

of

for

000

the of

with

determined

50 free

one

been

remain

the In

All

000

as

between

most

occur.

probably

20 These

particle,

and

proteins 000

they

1113

40s

G)

particles

fraction

the

weight

between occurs.

2 (track

additional -

bands of

1 (track

000

range

six

molecular

two

Mr

which

to

proteins

gradient

However and

weight

four

gradient

specific

simultaneously

the

of

from

weights

constituents

Additionally

In

from

000

lack

molecular

molecular

with

particle

identified. -

the

identical

particle

be

Heparin-sepharose C).

FG

series

H shows.

25-30s contain

can = 35

well

not

track

the K,L)

Mr

agree

certainly

only

polypeptides weights

E

a characteristic

are

000

I

D

SDS/polyacrylamide gel electrophoresis. The samples were electrophoresed 18% (A-D) and 10% (E-M) acrylamide slab gels. elF-3 (7 vg); its polypeptides were used as markers; their molecular weights, determined by Schreier et al. (131, are indicated in I. constituents of the gradient shown in Fig.1 25-303 particles (40 pg; fractions 39 - 46) 50s particles (fractions 24-30) after Heparin-sepharose chromatography; C: bound fraction (50 pg), D: non-bound fraction (30 ug) constituents of the gradient shown in Fig.2 205 particles (10 pg; fractions 26 - 28) 25-305 particles (20 ug; fractions 20 - 24) 40s particles (20 ~9; fractions 6 - II) free cmRNP (10 pg) free cmRNP after Heparin-sepharose chromatography; L: bound fraction (5 ug), M: non-bound fraction (5 ~9)

-

track

55

76 000

aid

of

(10; protein

BIOCHEMICAL

Vol. 99, No. 4,198l

and

polypeptides

ular

weights

with of

dominantly

ular

000

the

molecular weight

a strong

50 000

-

70

000

000

not

of

bound between

gradient

molecular

the the

weight

free

and

column

70

particles,

000

L).

It

20

molecpre-

Heparin-sepharose

50

the the

The

range

contains

the

to

000.

after

(track

respect

COMMUNICATIONS

n-sepharose

cmRNP

whereas

50s

2 with

000 Hepari

Likewise,

the M)

to

to

iJ).

(track

RESEARCH

50

bind

(track

fraction

exists from

between

do

proteins remain

similarity particles

> 30

non-bound weight

proteins

25-30s

which

to

BIOPHYSICAL

weights

proteins

20

chromatography 30 000

molecular

the

from

AND

the is

free 000

70

20 000

obvious

30

and

000

-

that

cmRNPs

-

000 molec-

the

and

the

polypeptides.

DISCUSSION The

200

riched

in

(9,lO).

000

In

60s

x g fraction

this

values

lower

of

particles

which

(Fig.l,2).

It

ticles

from

been

able

tested

the both

was

When

mRNA

260

nm in

the

40-50s

bility

of

the

50s

obvious: case

In of

are

of

this case

of

l-free

the protein

ever

complexes the

joining

the

peak

x

in

(Fig.21 gradient

and

the

200

the

50s

shown

prevents

reaction

the

000

during

Fig.1

the

50s

whereas

after

the

as

joining

reaction

also

occur,

must

been

excluded

incubation.

1114

much

mRNA

particles

are stabi

the

after of

complexes their

in

amounts

monosomes have

sta-

whereas

capacity

of

not

becomes

Moreover,

50s

yet have

at

equal

twice

translational

not we

absorbance

5OS,

405.

The

par-

a different

at

particles,

their

have

conditions

to

1).

we

the

compared,

fted

GTP-dependent

the

of

GuoPP(CH2)P

a consequence

x g fraction

from

coincide

(Table

80s

25-30s

2 (data

x g fraction

active

the

gradient

000

in as

synthesis,

hybridization

when

region

retain

25-30s

activity

shi

50s

of

However

peaks

the

with

prolonged

synthesis.

are

sed-

treatment

with

distribution

with

the

occurence

centrifugation

is

g fraction

20-30s of

200

two

of

with

synthesizing

gradients the

absorbance

translationally of

the

1 both

000

as

which

since

of

the

associated

particles the

gradient

the

gradient

GuoPP(CH2)P

in

200

centrifugation

from

and

under

of

2 the

region

protein

particles

[5’-3H]poly(U) is

amounts

considerably

in

by

which

25-30s

distribution

no

en-

synthesis

small

both the

result

protein the

regions

with

20-30s

in

of

particles case

the

associated

longed

ccl

the

gradient

treatment

active

but

is

protein

proteins,

and

shown

mRNA

demonstrate amounts

the

as

the

in

Strikingly,

g fraction

mRNA

1 is

comparative

shown).

x

that

similarly

(9,10,17).

lysates

active

ribosomes,

GuoPP(CH2)P

000

reticulocyte are

soluble

80s

40s

contain

shown

gradient to

contains

analogue

200

rabbit

which

inactive

than

GTP

centrifugation

the

and

nonhydrolyzable

from

complexes

fraction

subunits

imentation

the

50s

addition

ribosomal

the

isolated

mRNA-containing

mRNA pro-

occurs

in

derived (not

not

shown). active

I ization

from

in

with

(2,5,6,8). some

In

of

Howthe this

50s block

BIOCHEMICAL

Vol. 99, No. 4,19Bl

Their

similar

protein

sedimentation

patterns

cmRNP

which

weight

between is

absence

been

of

described

somal

mRNPs

the

patterns

only

be

tein

patterns.

50s

being

free

their

by

Due

to

complexes start the

osomal

are

(ii)

The

seems

taining

preinitiation

start Based

with on

the

initiation

depending

in

the

steps

of

active

80s

and

25-30s ribosomal

the

mRNP

76 from

patterns

and

two

types

of

mRNA

be

the

second

polyin

set

can

This

the

gel

par-

their

pro-

tracks

of

the

polypeptides

and

the

gradient

the

of

identified

to

single

000

has

Mr

-

1 (Fig.3B)

might

conditions

for

varied

25-30s

of

= 35 000

particles

have

been

allow

the

joining data

some

joining the

of

the

reaction. initiation

monosomes

or

particles

mRNA-containing which

sequence, in

which

Those

are

not

resulting

a decomposition is

preini-

into

presumably

in-

in 40s

dependent

eirib-

on

the

subunits.

increase

in

the

separation

centrifugation of

which

time

25-30s

had

not

of

particles been

the from

stimulated

200 the

by

an

000

x g

mRNA-conincubation

reaction. presented

sequence on

and -

particle

GuoPP(CH2)P

complexes

the

the

next

60s

to

that

blocked

considerable

fraction

protein

with

the of

of

complex.

000

the

can

by

mechanisms:

with

subunits

50s

different

formation

availability

25-30s

= 72

and

protein

regard in

proteins

Mr

the

propose

detectable

ribosomal The

incubation

hibited

also

molecular 70 000

2 (Fig.3G). with

the

free

This of

(Fig.3B,G)

compare the

the and

proteins

gradient

to

and

in

tract

of

identical

is

here

50 000

poly(A)

from

almost

us

(12,18-20).

set

COMMUNICATIONS

led

polypeptides

particles

pattern

the

content

between

the

first

particle thus

in

we

the

protein

25-305

with

different

tiation

ther

are

of

preparation,

76

with

(Fig.3C,D).

their

of and

000

RESEARCH

described

sets

the

25-30s

protein

constituents

generated (i)

the

cmRNP

observed

of

-

of

mRNA

30 000

associated

interspersed

Because

two

000

types

This

proteins be

= 72

and particles

and

Whereas

both

factors

also

the

as

in

the

particle,

38 000

by

(19,21,22).

initiation

to

Mr

recovered

and

25-30s

20 000

the

of

ticle

the

between

BIOPHYSICAL

values

characterized

ranges

the

AND

respective

the

it composition

is

conceivable of

its

(pre)inifiation

that

as

associated

complex

to

which

the

mRNA

traverses

proteins

may

it

presently

vary

belongs.

ACKNOWLEDGEMENTS

We express our appreciation to Drs. Kay I ene Edwards and stimulatory discussion and critical review of the manuscript. Lesyre for excellent technical assistance. This investigation the Deutsche Forschungsgemei nschaft within the SFB46.

Rai ner Hertel We thank‘Annette was supported

for by

Vol. 99, No. 4.1981

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Benne, R. B Hershey, J.W.S. (1978) J.Biol.Chem. 253, 3078 - 3087. Peterson, D.T., Merrick, W.C. B Safer, B. (1979) J.Biol.Chem. 254, 2509 - 2516. Safer, B., Adams, S.L., Anderson, W.F. & Merrick, W.C. (1975) J.Biol.Chem. 250, 9083 - 9089. Gross, M. (1979) J.Biol.Chem. 254, 2370 - 2377. Gross, M. (1979) J.Biol.Chem. 254, 2378 - 2383. Safer, B., Jagus, R. & Kemper, W.M. (1979) Methods Enzymol. 60, 61 - 87. Kramer, G., Odom, O.W. & Hardesty, B. (1979) Methods Enzymoi. 60, 555 - 566. Peterson, D.T., Safer, B. 8, Merrick, W.C. (1979) J.Biol.Chem. 254, 7730 - 7735. Sarre, T.F. & Hilse, K. (1978) Eur.J.Biochem. 82, 123 - 131. Sarre, T.F. & Hilse, K. (1980) Buhl, W.-J., Biochem. Biophys. Res. Commun. 2, 979 - 987. Rhoads, R.E. 8 McKnight, G.S. (1974) Schimke, R.T., Methods Enzyrrol. 30, 694. Jacobs-Lorena, M. 8 Baglioni, C. (1972) Proc.Nat.Acad.Sci. USA 69, 1425 - 1428. Schreier, M.H., Erni, B. & Staehelin, T. (1977) J.Mol.Biol. 116, 727 - 753. Laemmli, U.K. (1970) Nature 227, 680 - 685. Mumby, M. & Traugh, J.A. (1979) Biochemistry 18, 4548 - 4556. van der Mast, C., Thomas, A., Goumans, H., Amesz, H. 8 Voorma, H.O. (1977) Eur.J.Biochem. 75, 455 - 464. Buhl, W.-J., MiGa, T., Sarre, T.F. & Hilse, K. (1977) Hoppe-Seyler's Z.Physiol.Chem. 358, 1187. van Venrooij, W.J., van Eekelen, Z.A.G., Jansen, R.T.P. & Princen, H.M.G. (1977) Nature 270, 189 - 191. Van-Tan, H. & Schapira, G. (1978) Eur.J.Biochem. 85, 271 - 281. Princen, H.M.G., van Eekelen, Z.A.G., Asselbergs, F.A.M. & van Venrooij, W.J. (1979) Mol.Biol.Rep. 2, 59 - 64. Blobel, G. (1973) Proc.Nat.Acad.Sci. USA 70, 924 - 928. Schwartz, H. & Darnell, J.E. (1976) J.MoI.Biol. 104, 833 - 851. Anderson, W.F., Bosch, L., Cohn, W.E., Lodish, H., Merrick, W.C., Weissbach, H., Wittmann, H.G. & Wool. I.G. (1977) FEBS Let-t. 76, 1 - 10.

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