Physicochemical characteristics of isolated 55-S mitochondrial ribosomes from rat-liver

Vol. 54, No. 1, 1973

BIOCHEMICAL

AND BIOPHYSICAL

PHYSICOCHEMICAL CHARACTERISTICS

RESEARCH COMMUNICATIONS

OF ISOLATED

55-S MITOCHONDRIAL

RIBOSOMES FROM RAT-LIVER Hans de Vries Laboratory

Received

July

and Rya van der Koogh-Schuuring

of Physiological Chemistry, State University, Bloemsingel 10, Groningen, The Netherlands

13,1973

SUMMARY A procedure for the preparative isolation of rat-liver mitochondrial ribosomes free of cytoplasmic contamination is described. The 55-S ribosome was characterized by electrophoretic analysis on acrylamide and agarose gels, It is concluded that its charge/mass ratio is very low and that its volume is larger than that of E.coZi ribosomes and smaller than that of rat-liver cytoplasmic ribosomes. Therefore, the 55-S ribosome is not adequately described by the term "miniribosome".

INTRODUCTION During

the last

years,

contain

small

mitochondria as the native

the sedimentation

tain

(for

reviews,

into

the

(sometimes structures.

smaller

presented real

accepted called

in

dimensions

size

its

the small

cytoplasmic

paper

were

RNAs they the

counterpart

meant

and physicochemical

animal

was deduced

Electronmicroscopically

than this

that

"miniribosomes")

The small

55 S, and from

I and 2).

refs.

somewhat

The experiments

55-S

ribosomes

coefficient, see

appears

insight

has become generally

protein-synthesizing

from

particle

it

con-

55-S 3

.

to get a better

properties

of the

ribosome.

METHODS a. Buffers: threitol, acetate, 0.25

KMEDT buffer: 10 mM Tris-HCl,

100 mM KCl, pH 7.6;

6 mM f3-mercaptoethanol,

M sucrose,

b. Isolation

1 mM Tris-HCl,

10 mM MgC12,

Gel buffer:

60 mM NH4 acetate,

10 mM Tris-acetic

acid,

1 mM dithio4 mM Mg

pH 7.6;

ST buffer:

pH 7.4.

and washing of ntitochondria:

Copyright @ 1973 by Academic Press, Inc. AN rights of reproduction in any form reserved.

0.1 mM EDTA,

308

The method

has been

adapted

BIOCHEMICAL

Vol. 54, No. 1,1973

rats

of go-120

throughout. 3,200

. For each experiment

g, starved

After

overnight,

and spun down at

suspended

cells

After

added

were

19,000

(giving

werespundown

The final

at 19,000

pellet

was suspended

suspended

20 % Triton 9,500O

and lysed

x g for

stirring

IO min,

X-100

rotor,

After

in KMEDT buffer.

the pellets,

in KMEDT buffer,

containing

layered

a flow-cell

der was connected, fractions

were

and the peak

in a Beckman Ti50

twice

rotor.

with

IO ml at I M sucrose,

resuspended in the same

and spun

collected.

for

were

15 h at

pumped

to which

a recor-

The ribosomal

and spun for 4 h at 50,000rpm

KMEDT buffer

The pellets

were

gradients

254 nm),

were

of

rpm in a Beckman

ribosomes

(wavelength

fractions

mitochondria

IO ml of

at 4’ C. The gradients

of a LKB Uvicord

diluted

onto

% (w/v))

rpm in a Beckman SW27 rotor

washed

C and clarification

the

22,000

c.

by the addition

sucrose

15.0

per

protein).

under

3 h at 60,000

top

were

and spun down again.

as described

on 35 ml isokinetic

(sucroseconcentrationat

through

resuspended

mitochondrial

buffer

resus-

to 1000 ml and the mitochon-

was layered

After

x g),

the mitochondria

mg digitonin/mg

5 min at 0’

the supernatant

(I min at

1.75 mg of digitonin

and lysed

for

was used

removed

from mitochondria:

140 ml KMEDT buffer

X-100.

I % Triton Ti60

in

Then

- 0.16

5 min,

ST buffer

were

was diluted

c. IsoZation of ribosomci! particles were

Sterile

containing

x g for

of 40 male Wistar

livers

(2 min at 17,000

5 min.

of 0.12

2 min at 0’ C, the suspension

used.

pelleted

x g for

a ratio

the

RESEARCH COMMUNICATIONS

and nuclei

and 200 ml buffer

in 200 ml,

ml were

were

homogenization,

x g) and the mitochondria

pended

dria

4

et al.

from Loewenstein

AND BIOPHYSKAL

of ribosomal

particles

were

resuspended

in KMEDT buffer. d. Other ribosomes: Total according

to Blobel

ribosomes

by sucrose-gradient

Total

B.coli

ribosomes

70-S

ribosomes

were

rat-liver

& Potter5;

80-S

ribosomes

ribosomes were

isolated

were

prepared

from

these

separation.

and S-100 purified

cytoplasmic

were

prepared

on sucrose

309

gradients.

according

to Nirenberg6.

Vol. 54, No. 1, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS AND DISCUSSION

Sedimentation characteristics Initially, without with

rat-liver

puromycin lysosomal

enzymes,

ethylacetate-soluble

this,

the digitonin method

ting

oxidative

Fig.

1.

mitochondrial

in the peptidyl

small

this

of mitochondria ribosomes.

causing

transferase splitting

molecules treatment

effectively

ribosomes

reaction'. off

from

lysosomes

phosphorylation4.

Fig.

very

high

Since

or other

acetyl-leucyl-tHNA, (cfi

might

explain

METHODS) was introduced:

from mitochondria

1 shows

blanks

contamination

of acetyl-leucine

of mitochondria

removes

showed

without

the profile

that

affec-

was obtained

Separation of mitochondrial ribosomes on isokinetic sucrose gradients. The procedure is described under METHODS. Sedimentation coefficients were relative to N.crassa cytoplasmic 77-S ribosomes run in a parallel tube.-: ribosomes from mitochondria washed with digitonin; - - -: ribosomes from mitochondria washed twice with 0.25 M sucrose, 1 ti EDTA, pH 7.4.

when crude

mitochondrial

gradients.

Mitochondria

of 56-S peaks

ribosomes

(about

ribosomes

were

treated

with

30

A260 units

of 39 S and 28 S. A clear

separated

digitonin

difference

310

on isokinetic yielded

from

40 rat

sucrose

a major livers)

was seen with

fraction

and subunit ribosomes

from

BIOCHEMICAL

Vol. 54, No. 1, 1973

mitochondria with

washed

this

method

the digitonin but

also

without

treatment 80-S

quite

that

0.25

M sucrose,1

peak of 82-S

ribosomes

does not only

remove

were

indeed

lysosomal

brought treated

hydrolases

mM EDTA instead was obtained. lysosomes

The latter

In the peptidyl

from mitochondria

assumption

with

ribosomes.

by Malkin8.

puromycin

ribosomes

assay

a high

adhering

observations

twice

AND BIOPHYSLCAL RESEARCH COMMUNICATIONS

finding

transferase

is

Obviously, the mitochondria

consistent

reaction

back

to normal

in this

way were

interfere

from

of digitonin:

with

with

the blanks

values

if

55-S

used.

This

makes the

the peptidyl

transferase

plausible.

EZectrophoresis of nntochondriaZ ribosomes. Fig.

2a shows

a comparison

0.75 0 r

A

of the migration

velocities

of 55-S ribosomes

@I

A

A260

;; ii

i iI\

0123156

Fig.

2.

0123456 distance

migrated

(cm)

Gel e2ectrophoresi.s of ribosomes. A: 2.2 % polyacrylamide gels. The electrophoresis was performed according to Talens et al.9 , except that pre-swelling was omitted. The buffer was 74 mM NH4 acetate, 5 mM MgCl , 50 s&l Tris-acetate, pH 7.8. Electrophoresis was for 3 h at 12 Ini/ gel at 40 C. B: 0.4 % agarose gels. The electrophoresis was carried out according to Talens et aZ. 10, in gel buffer (METHODS). Electrophoresis was for 2.5 h at 9 mA/gel at 4O C. Gels were scanned at 260 nm on a Gilford 2400-S spectrophotometer with a linear transport system. -: 55s; -a-+: rat-liver 805; ---: E.coZi 70s.

311

BIOCHEMICAL

Vol. 54, No. 1, 1973

and of rat-liver

cytoplasmic

acrylamide

As expected’,

gels.

the smaller of running not

in

front

allowed,

the 55-S reason

ribosome this

gels

2b).

Under

2b shows

80-S

ribosomes

ribosomes

have

probably

Fig.

like

a very

low negative

between

it

suggests

ments

1

has also

of 55-S

may also 70-S,

ribosomes

been

(that found

ribosomes

in get

to its

is much higher

in

although

its

penetration. 80-S

acrylamide

and

gradients.

intermediate split

et al. “lo). than

to

influence

a position

RNA.

the acryl-

migrated

ribosomes

70-S

which

to a point

cytoplasmic

in

55-S

to its

of a ribosome,

3-8 % linear

by Talens

mass,

ribosomes

rat-liver

E.coZi

that

relative

slightly

stuck

fastest,

indicates

a gradient

on the volume

ribosomes

ribosomes

with

ratio.

travel

IO,1 I . The distance

possible

effect

This

up into shows

sedimentation

that

coefficient

.

These 260

the 55-S

protein

to electrophorese

of E.coZi 55-S

as compared

can

% agarose

by the charge/mass

(i.e.

depends

the migration

two populations the volume

is

This

in 0.4

This

of

ratio

no sieving

slowest.

is

Another

ribosomes.

70-S ribosomes

gradient

and flexibility

and 80-S

of other

of ribosomal

is possible

therefore

that

70-S

size

penetration

evident

charge/mass

the

mobility

charge

amount

a pore

mitochondrial

is

an A

ribosomes

rigidity

3 shows

rat-liver It

and 55-S

the volume

be that

determined

slower

80 S. It

ribosome.

practically

conditions

equilibrium

factors

only

than

that

than

instead

of the 80-S

than

circumstances is

under

concentration)

reach

lower

these

with

no further

could

move slower

slower

by extrapolation that

on 2.2 % poly-

ribosomes,

considerably

than

is

these

due to a high

In gels

that

that

ribosomes

the electrophoretic

velocity

Fig.

80-S

55-S mitochondrial

behaviour

ribosome

ribosomes

70-S

larger

to conclude

by investigating

(fig.

amide

but

unexpected

occurs,migration

is

the

is much larger

of the mitochondrial be tested

and E.coZi

of 70 S, migrate

of course,

for

80-S

ribosomes,

E.coli

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

experiments

nm’A230 , causing

were

done with

nm ratio

of

a slow

migration

highly

1.2 or higher. velocity

312

purified

55-S

Contamination in gels,

could

ribosomes

with

membrane

be excluded

showing fragwith

BIOCHEMICAL

Vol. 54, No. 1, 1973

r..

I

vo

*.

*

2 4 of electrophoresls

time

Fi .g.

AND BIOPHYSICAL

*

*

6

RESEARCH COMMUNICATIONS

J 3%

6 (days1

Electrophoresis of ribosomes onc pore size gradient of poZyacrylamide. Linear 3-8 % polyacrylamide gradients were made was in “gel as described by Talens et aL. 10. Electrophoresis at 3.6 mA/gel at 4’ C, with continuous circulation of buffer”, the buffer via a peristaltic pump. Runs were in triplicate, and 55-S ribosomes were from two separate experiments. At the times indicated gels were scanned (see fig. 2), and then electrophoresis was continued. o---o: 55s; -: rat-liver 80s; Ad: E.coli 70s.

3.

the

certainty: pholipids

ribosomes

from mitochondria

by [‘4C]-ethanolamine

were

labelled

the only

in

viva

fractions

in their

definitely

phosdevoid

of radioactivity. It

is possible

to make a rough

estimate

we have determined

the buoyant

1.43.

to 37 % RNA and 63 % protein

This

similar

accords

low density

content

RNA

per

has also

ribosome

of the 55-S ribosome

E.coZi

ribosomes15.

very

rough

ribosome

that

is

those

evident

chloramphenicol-sensitivity

(cf.

1 .O x IO6 D (cf. about in the

2.7

ribosomes

of 70-S

arrive and 80-S

the ribosome

We now interpret

of the peptidyltransferase

313

volumes: ribosomes

also

fig.

ref.

about exist

to be

2b).

A

13 , Taking

and Ojala t4),

x 106, equal

gel buffer

we again

that

as a 55-S particle.

I2

by Attardi

of different

between it

in CsCl of the 55-S

found

then

of course),

intermediate

can be isolated 16

densities

assumption,

In conclusion,

is

Assuming

the buoyant

been

as about

weight

between

density

of the ribosomal

the

the molecular to that

the

of

same ratios

as in CsCl

at a volume

of the

(a 55-S

ribosomes. of rat-liver the previously reaction

mitochondria reported of

80-S

Vol. 54, No. 1, 1973

particles

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to be due to contamination

Our results in

BIOCHEMICAL

further

show that

and the low sedimentation

mitochondrial weight.

Its

low charge

ribosome

is not

protein

with

“mini”

with

respect

are

55-S particles,

the “mini-RNAs”

of the 55-S

properties

high

fraction

notwithstanding

coefficient

physicochemical and its

of this

ribosome,

the rat-liver

to volume

determined

present

and molecular

by its

relatively

content.

ACKNOWLEDGEMENTS We wish Prof.

F.J.

to thank Loomeijer

manuscript

of ref.

generously

given

Netherlands from

for

A.M.

Kroon

for

encouragement

10 available by Pfizer

Foundation

the Netherlands

Dr.

Ltd.

and Prof.

to us prior This

of Chemical Organization

criticism

and helpful L. Bosch

to publication.

work

was supported

Research

(S.O.N.)

for

the Advancement

suggestions,

for

making Anisomycin

in part with

the

by the

financial

of Pure

was

aid

Research(Z.W.O&

REFERENCES 1. Borst,P. and Grivell,L.A., FEBS Letters, 13, 73(1971) 2. Kroon,A.M., Agsteribbe,E. and De Vries,H., in “The Mechanism of Protein Synthesis and its Regulation”, Ed. by Bosch,L., North-Holland, Amsterdam, 539(1972) 3. Aaij,C., Nanninga,N. and Borst,P., Biochim.Biophys.Acta, 277, 140(1972) Scholte,H.R. and Wit-Peeters,E.M., Biochim.Biophys.Acta, 4. Loewenstein,J., 223, 432(1970) 5. Blobel,G. and Potter,V.R., J.Mol.Biol., 26, 279(1967) in Enzymology”, vol.VI, Ed. by Colowick,S.P. 6. Nirenberg,M.W., in “Methods and Kaplan,N.O., Academic Press, New York, 17(1963) 7. De Vries,H., Agsteribbe,E. and Kroon,A.M., Biochim.Biophys.Acta, 246, 111(1971) 48, 1106(1972) 8. Malkin,L. I., Biochem.Biophys.Res.Commun., Kalousek,F. and Bosch,L., FEBS Letters, 12, 4(1970) 9. Talens,J., Van Diggelen,O.P., Brongers,M., Popa,L.M. and Bosch,L., 10. Talens,J., Eur. J. Biochem., in press II. Caton,J.E. and Goldstein,G., Anal.Biochem., 42, 14(1971) 12. Perry,R.P. and Kelley,D.E., J.Mol.Biol., 16, 255(1966) 13. Attardi,G. and Ojala,D., Nature New Biol., 229, 133(1971) 14. Borst,P. and Flavel1,R.A.. FEBS Symp., 28, l(1972) Synthesis and its Regulation”, 15. Spirin,A.S., in “The Mechanism of Protein North-Holland, Amsterdam, 515(1972) Ed. by Bosch,L., Van der Koogh,H.J.L. and Kroon,A.M., 8th FEBS Meeting, 16. De Vries,H., Amsterdam, 1972, abstr. 545

314