Completion of nascent hela ribosomal proteins in a cell-free system

Vol. 40, No. 1, 1970

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

COMPLETIONOF NASCENTHELA RIBOSOMALPROTEINSIN A CELL-FREE SYSTEM Judith

E. Heady and Edwin H. McConkey

Department of Molecular, Cellular and Developmental Biology University of Colorado Boulder, Colorado 80302

Received

May 14, 1970

SLIMMARY. HeLa cell 3H-labeled proteins completed in vitro and 14C-labeled 60s subunit ribosomal proteins isolated from cells labaedvivo were processed -Alinear gradient together and applied to a carboxymethyl-cellulose column. of NaCl was used to elute the adsorbed proteins. Prominent peaks of both isotopes eluted together at O.lM and from 0.175M to 0.25M NaCl. Polyacrylamide gel electrophoresis of the doubly-labeled proteins confirmed the identity of the ribosomal proteins completed --in vitro with the ribosomal proteins labeled in vivo. -We wish to determine synthesis.

Although

in the nucleolus

it

(l),

is well

that

at least

plasmic

(2,3),

is still

some nuclear

a cell-free

of protein

proteins

synthesis

polypeptides.

We have adapted their

tion we report

preliminary

within

(4); but it

Gallwitz

permits

molecules

of rP synthesis

- the histones

(5,6).

system that

site

protein

(rP)

RNA (i-RNA) is synthesized

rRNA precursor

controversial

polysomes in HeLa cells

reported

join

of ribosomal

ribosomal

the actual

and even the existence

most types of cells

site

known that

and that rP's

RNA's leave the nucleolus The extent

the intracellular

before

is unknown.

the nuclei

is virtually

the completion

on cytohave

of nascent histone

system to our needs.

evidence for the cell-free

(6,7)

of

certain

- are synthesized and Mueller

the

In this

completion

communicaof nascent

HeLa rP's. METHODS. HeLa cells Essential

grown in suspension

were

Medium (8) supplemented

To prepare marker rP's, free medium, to a final

with 5% calf

a cell

concentration

culture

culture

Minimum

serum. was diluted

of 2 x lo5 cells/ml 30

in Eagle's

1:l

with amino acid-

and a mixture

of 15

Vol. 40, No. 1, 1970

14C-amino acids uCi/ml final

(uniformly

labeled,

concentration.

After

and ribosomal Malville

subunits

and Eldridge The cells

(Sorvall).

leted

were swollen

by overnight

(A)l,

concentration

over buffer

followed.

(C)l and

(C) with puromy-

Next, MgCl, and KC1 were

(D)l and the subunits

were separated

on

(D).

the procedures

of Gallwitz

and Mueller

However, an Omni-Mixer was used to break the buffer

to 2.2 mg of microsomal protein

made with O.OOlM Dithiothreitol

was for 20 minutes at 37'.

(DTT).

Up

per 0.5 ml and 0.6 to 0.8 mg pH 5.0 fraction

per 0.5 ml were used (measured by a modification

hydrolyzed

from

at 10,000 g for 10 minutes.

of 5 x 10s4 M.

in buffer

and

and the ribosomes were pel-

(B)l

of 10 to 30% sucrose in buffer

in the incorporation

The supernatant

at 37' for 30 minutes in buffer

To complete nascent rP's in vitro,

England Nuclear)

of 0.3M and the nuclei

These were resuspended in buffer

concentration

added to the concentration

cells

of McConkey, Linsky,

at 15,000 g for 15 minutes.

Ribosomes were then incubated

(6,7) were closely

ribosomes

and broken in an Omni-Mixer

were removed by centrifugation

gradients

were harvested;

by the procedures

in buffer

centrifugation.

added to a final

was added to 0.75

(9) and Linsky and McConkey (10).

low-speed spin was layered

isokinetic

24 hours the cells

were prepared

were pelleted

clumps and dirt

tin

New England Nuclear)

KC1 was added to a final

mitochondria this

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3H-amino acids

of Lowry, 11).

(randomly labeled

at 150 uCi per 0.5 ml were used as label.

with l.OM NaOH for 15 minutes at room temperature

Incubation

mixture,

New

Charged tRNA was before

samples

were counted for radioactivity. The mixture ribosomal The pellet

of 3H-labeled

subunits

proteins

was precipitated

in 10% trichloroacetic

was washed twice with ether,

(A) O.OlM (DTT). (B) 1.75M O.OOlM Dl-T. (C) 0.05M (D) 0.05M

Tris-HCl

pH 7.4,

pH 7.4, pH 7.4,

incubated

and the 14C-labeled acid for 30 minutes.

for 60 minutes at 37' in

O.OOlM MgC12, O.OlM KC1 and O.OOlM Dithiothreitol

sucrose in 0.05M Tris-HCl Tris-HCl Tris-HCl

completed -in vitro

pH 7.4,

O.OOlM MgClp, 0.08M KC1 and

O.OOlM MgCl,, 0.025M KC1 and O.OOlM DTT. O.OlM MgCl,, l.OM KC1 and O.OOlM DTT. 31

BIOCHEMICAL

Vol. 40, No. 1, 1970

ethanol-ether pellet

(1:l)

and this

pellet

was resuspended and left

for several 15 minutes.

hours at 0'.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was again washed twice with ether.

in 6.OM guanidinium

The insoluble

The supernatant

rpm for 60 hours to separate

material

chloride

was layered

over 4.75M CsCl and spun at 40,000

the nucleic

acids from the protein.

and dialyzed

(6.OM urea,

O.OlM sodium acetate

filtered

(12),

mine (12) and 0.1% 2-mercaptoethanol The mixture

of labeled

of carboxymethyl-cellulose adsorbed proteins

was kept constant

tate was collected

gel electrophoresis,

10% trichloroacetic

and the protein

with 0.05M sodium acetate

Details

were collected

buffer,

buffer,

0.1% methyla-

Whatman).

gradient

The

of 0 to 0.35M the flow

pump. peak fractions

from the column

(BSA) was added to each and they acid for 30 minutes.

was eluted

pH 5.0.

gel at pH 4.5 (13,14)

on a 0.9 x 13 cm column

every 10 minutes;

on a 0.45 u pore size Millipore

small pieces

polyacrylamide

linear

at 12 ml/hr with a peristaltic

with

the column buffer

CM 52, preswollen,

200 pg of bovine serum albumin

were precipitated

cut into

was fractionated

with a 1 liter

Fractions

For polyacrylamide were pooled,

proteins

were eluted

against

The fractions

at pH 5.6) for 24 hours.

(CM-cellulose;

NaCl in column buffer.

(GuCl), pH 5.6,

was removed at 15,000 g for

with OD2ao/OD2sa > 1.2 were pooled, charcoal

The

filter.

The precipi-

The filter

was

with 0.25 ml of 8.OM urea

Each eluted

fraction

was run on a

or pH 8.7 (15) toward the cathode.

appear in the legend of Figure 2. The gels were crushed with a Savant gel crusher

1.0 ml of distilled samples to a final 37' in tightly

water as diluent. concentration

capped vials.

in a Beckman LS-133 scintillation

Thirty

percent

ing the total

proteins

chains

HZ02 was added to the

of 3%; samples were incubated The fractions

overnight

at

were counted in EGME-butyl PBD2

counter.

RESULTSAND DISCUSSION. To avoid uncertainties completed polypeptide

at 2 mm per sample with

from polysomes,

of the incorporation

2

about the release

we developed mixture

a method for prepar-

for CM-cellulose

2 ethoxyethanol: 2-(4-tert-Butylphenyl)-5-(4-Biphenylyl-l,3,4-Oxadiazole (8 gm/liter in toluene) [l:l] 32

of

Vol. 40, No. 1, 1970

chromatography. Kurland, never

Initially,

Voynow able

to recover however.

extracted

proteins

by weight),

bridge

of

the

lipids

with

total

50

works

well

25% of

the

density the

acid for

(16), developed

100

125

for

150 COLUMN

175

200

FRACT0N

Figure 1. CM-cellulose column chromatography and 3H-labeled proteins completed in vitro: cpm of 4 of every third fraction. The ratio 45. Recovery was 75% for the 3H and 50% for 33

contaminants.

the

acid(2 3.5% In addition,

to prevent

disulfide

columns. laevis

the

identification

225

acetic

contaminants

Xenopus

on CM-cellulose

We were

CM-cellulose

these

acid

efforts

from

with

a method

proteins

of

the

(9).

from

of nucleic

recovery

of Hardy,

HeLa rP’s

ratio

and careful

working

extraction

radioactivity

280/260

presence

to improve

cell

75

acetic

ethanol-ether

and Brown cells,

the

RNase nor DNase removed

failed

of

*5

than

indicated neither

kidney

presence

which

The optical

formation

cultured

(12)

more

but

Hallberg

we tried

and Mora

columns,

removal

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

eggs,

columns.

250

275

embryos

and

of

in

rP’s

A crucial

300

325

step

350

NUMBER

of HeLa cell ‘?-labeled rP’s Each point on the graph represents of 3H/14C put on the column was the 14C.

Vol. 40, No. 1, 1970

in their

BIOCHEMICAL

method

was solubilization

overnight

incubation

to

their

employ

e.g.

those

in

involving

lipids

subunit

were

aspartyl

least

so that from

red

the

Figure

1 shows

and 0.25M

at O.lM

ratio

three

not

of the

those of the

3H-labeled

we demonstrate

the

CM-cellulose

these adsorbed

of

laevis

the

subunits to

the

the

of the

eluted

(3H)

between

gel

provide

14C rP’s

with

occur-

additional

proteins

is

attested

the

possibility

that

elution

profile

of the

3H/14C Figure

evidence

that

In Table in

the

gel

Apparently,

sample.

14C marker

The use of

lowest

identical.

almost

active

3H-labeled

electrophoresis.

are

3H in the

are

the

3H and 14C labels

of the

among the

rP’s

(10).

--in vitro

3H and 14C labels

peaks

acrylamide

and the

above

at

1

patterns some

exclusively. to by the

fact

that

in the.poly

U-dependent

syn-

1M KC1 in the

preparation

of

non-ribosomal

proteins

were

ribosomes. that

resemble rP’s

of

contained

contamination.

were

contain

as described

minimizes

rP’s

and

or four

these

represented

which

of

Three

completed

the

60s

in Methods

CsCl.

fractions

polyphenylalanine

We note closely

of

of

and the

NaCl.

correspondence

column

The validity

thesis

of proteins

to

fraction

the

acid

actually

patterns,

as explained

1.25 % nucleic

nine

proteins

proteins

that

prepared

were

bonds,

After

mixture

pooled;

correspondence

rP’s

unwilling

separation.

were

All

0.25M

gel

due to a trivial

subunits

than

to

by

of some peptide

density

enter

gradient

and concentrated

and subjected

some of the

is

whether

we pooled

2 presents

0.175M

We were

lability

CsCl

would

(14C).

The best

37’.

incorporation

over

profile

60s subunit

To determine proteins,

acids

an elution

and from

of the

layered

and less

NaCl.

known

use of buoyant

proteins

GuCl,

at

contaminants

(17).

35% of each

protein

the

of the

to the

nucleic

upper

the

from

in

acid

residues

the

the

80% of

and rP’s O.OlM

removed,

dispersed

centrifuged fractions

because

we turned

were

of phosphorus-containing

10% trichloroacetic

technique

Therefore, the

AND BIOPHYSICAL RESEARCH COMMUNKATIONS

or the

the the

patterns

patterns

published published

60s rP’s

by Hallberg by Kanai

34

--et al.

in Figure

1 does

and Brown

(16)

(18)

rat

for

for liver

not -X. and

Vol. 40, No. 1, 1970

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE 1

Input 3H cpm/14C

Recovery 3H cpm/14C

cpm; ratio

cpm; ratio

6860/1030

; 6.66

4867/939

; 5.2

1920/570

;

3.37

1380/396

; 3.48

5060/1680

;

3.01

2689/1258

; 2.14

I Recovery of 3H of selected CM-cellulose determinations, a 20 on each gel was mixed diluent and incubated

and 14C from acrylamide gel electrophoresis column fractions. For the input nl sample of each column fraction placed with 2 mm of blank gel and 1 ml of in the same manner as the gel fractions.

1400-

1200

300--300

T ,I I EJ 1

j.

T

g

x

t 0 200 -- 200 ‘.

% CJ -

1000

BOO :

ii,

5 GEL

FRACTION

NUMBER

Figure 2. Gel electrophoresis of regions (a), (b) and (c) of Figure 1. (a) and (c) were run at pH 8.7 toward the cathode for 6% hours at 3.0 ma/gel. (b) was run at pH 4.5 toward the cathode for 3 hours at 2.5 ma/gel. All of the fractions not appearing on the graphs were at background level for both isotopes. 35

Vol. 40, No. 1, 1970

skeletal

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

This could be due to differences

muscle rP's.

Finally, cytoplasmic

our results localization

can be interpreted of rP synthesis.

in technique.

as tentative This matter

evidence

for the

is now being investi-

gated more thoroughly. ACKNOWLEDGEMENTS This research NIH HD-02282.

J.E.H.

was primarily

supported

is the recipient

by NSF

GB-8009,

of an NSF predoctoral

and in part by traineeship.

REFERENCES 1.

2. 3. 4.

5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Perry,

R. P. In "Progress in Nucleic Acid Research and Molecular Biology", Vol. 6. (J. N. Davidson and W. E. Cohn, eds.) Academic Press, New York, p. 220 (1967). Warner, J. R. and Soeiro, R. Proc. Nat. Acad. Sci. U.S., z, 1984 (1967). Liau, M. C. and Perry, R. P. J. Cell Biol. 42, 272 (1969). Goldstein, L. In "Advances in Cell Biology", Vol. 1 (D. M. Prescott, L. Goldstein and E. H. McConkey, eds.) Appleton-Century-Crofts, New York, p. 187 (1970). Robbins, E. and Borun, T. W. Proc. Nat. Acad. Sci. U.S., 57, 409 (1967). Gallwitz, D. and Mueller, G. C. Science, 163, 1351 (1969). Gallwitz, D. and Mueller, G. C. European rBiochem., 2, 431 (1969). Eagle, H. Science, 130, 432 (1959). McConkey, E. H., Linsky, L. F., Malville, N. K. and Eldridge, D. Manuscript in preparation. Linsky, L. F. and McConkey, E. H. Manuscript in preparation. Bailey, J. L. "Techniques in Protein Chemistry", 2nd Ed. Elsevier, New York (1967). Hardy, S. J. S., Kurland, C. G., Voynow, P. and Mora, G. Biochemistry, 8, 2897 (1969). Reisfeld, R. A., Lewis, U. J. and Williams, D. E. Nature, 195, 281 (1962) Leboy, P. S., Cox, E. C. and Flaks, J. G. Proc. Nat. Acad. Sci. U.S., 52, 1367 (1964). Davis, B. J. Ann. N. Y. Acad. Sci., 121, 404 (1964). Hallberg, R. L. and Brown, D. D. J. Mol. Biol., 46, 393 (1969). Light, A. In "Methods in Enzymology", Vol. XI (C. H. W. Hirs, ed.) Acad. Press, New York, p. 417 (1967). Kanai, K., Castles, J. J., Wool, I. G., Stirewalt, W. S. and Kanai, A., FEBS Letters, 5, 68 (1969).

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