Mitochondrial membrane organisation, a determinant of mitochondrial ribosomal RNA synthesis

Vol. 43, No. 2, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MITOCHONDRIAI MEMBRANE ORGANISATION, A DETERMINANT OF MITOCHONDRIAL RIBOSOMAL RNA SYNTHESIS. I.

T. Forrester,

Department

Received

March

K. Watson

and Anthony

W. Linnane.

of Biochemistry, Monash University, Victoria, 3168. Australia.

Clayton,

16, 19’71

isolated from lipid-supplemented anaerobically SUMMARY: Mitochondria grown Saccharomyces cerevisiae contain normal mitochondrial ribosomes while the mitochondrial precursor particles isolated from anaerobic cells, depleted of both unsaturated fatty acid and ergosterol appear to lack mitochondrial rRNA. These findings indicate that the control of mitochondrial rRNA synthesis is directly related to the composition and molecular structure of the mitochondrial membrane. The morphology grown

Saccharomyces

composition contain

only

which,

in

primitive

turn,

lipid

mitochondrial

Watson

et al.

supplemented

vitro -*

well

quite

distinct

yeast

cell

It

the

This

to be a major synthesis.

factor

that

reports

architecture control

mitochondrial

rRNA while

acid

mitochondrial

rRNA and functional

ribosomes. 409

growth

from

by lipid-

incorporating precursor

structures

activity

lipid

composition

of mitochondrial

lipid-supplemented

the

development

mitochondria

incorporating

cells

in

has been reported

of the mitochondrial

The lipid-depleted the

system

the

lipid-

those

on the

mitochondrial

that

of lipid

of anaerobic

whereas

acid

lipid

structures

from

medium

amino

lipid-depleted

in the

mitochondrial

synthesizing

amino

whereas

The effect growth

by the depleted

structurally

have an active

communication

molecular

defined

was found

anaerobes

Cells

structures

of the

protein

of anaerobically

affected

2).

(1,

(1).

to have negligible

therefore

(rRNA)

possess

(3).

in vitro,

is markedly

medium

composition

of the

properties

mitochondrial

are

grown

appeared

growth

cells

aerobically

system

cerevisiae

of the

supplemented

and the

and biochemical

membrane ribosomal

appear cells

-in and appears RNA

to lack contain

normal

Vol. 43, No. 2, 1971

All

MYEYCHODS

BIOCHEMICAL

experiments

diploid

strain

basic

0.5% Difco

source

for

ethanol

yeast

anaerobic

--et al.

grown

yield

anaerobic

in

cells (E);

cells

galactose-yeast

aerobically a normal

stationary

phase if

viable; cells

the

begin

by Lamb et al. sorbitol

gradient

and cytoplasmic dodecyl ergosterol base

sulphate

centrifugation

as previously

Yeast

mitochondria

diethyl

as described

for

410

high

cells

grown

aerobic

cells

at early

than

90%

some hours

this

the

work yeast as described by discontinuous

total

mitochondrial

RNA

pyrocarbonate-sodium

(5).

Fatty

by Jollow

of Katz

contain

4%

were harvested

were isolated

described

by the method

anaerobic in

were purified

using

grown

supplemented

to yield

In

(1) and the

rRNA extracted

cells

denotes

are left

The mitochondria

(UFA)

E to yield

shown to be greater

ribosomes

acid

E deficient

and must be discarded.

were determined

compositions

RESULTS

(4).

medium,

cells

cultures

and cytoplasmic

with denotes

medium

All

to

Tween 80 and E, to yield

extract

deficient

medium

fatty

(e) Aer-EtOH

and the population

to die

mitochondria

cells;

denotes

grown anaerobically

An-Gal+T

containing

used

(a) An-Gal

grown anaerobically

composition.

lipid

cells

to yield

cells

whereas

extract

extract

1% ethanol-yeast

lipid

thus

supplemented

of UFA's

anaerobic

in

(4$),

unsaturated

denotes

medium,

The carbon

The nomenclature

both

medium

denotes

extract

lipid-supplemented

with

in

in UFA; (c)

as a source

(1).

4% galactose-yeast

extract

(d) An-Gal+T+E

cells;

medium

isolated

M, grown on a

was galactose

in 4% galactose-yeast

Tween 80,

a locally

strain

been adopted:

(b) An-Gal+E

deficient

with

growth.

deficient

in 4% galactose-yeast

anaerobically

growth

(1) has also

and ergosterol

anaerobic

- salts

aerobic

anaerobically

out

cerevisiae,

extract

(1%) was used for

cells

with

were carried

of Saccharomyces

strict

by Watson

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

acids

et al.

and

(6),

RNA

and Comb (7). molecular

weight

RNA species

BIOCHEMICAL

Vol. 43, No. 2, 1971

quite the

distinct, rest

both

in basic

cell

(5,8,9,10,11).

of the

components

derived

from

of the mitochondrial identified

with

aerobically

grown,

species

from inated

with

the

faster

in Figure

An-Gal+T+E

sucrose

density

mitochondrial drial

the An-Gal

mitochondria

unsaturated

fatty

saturated

lower 2.5

than mg/g

structures but

instead

sedimentation the

acids

which

mitochondria. is

dry

that

(Figure

0.4

mg/g

found

for

An-Gal+T+E

a prominent coefficient

two cytoplasmic

dry weight

contained

of 6-14s

rRNA species

which

from

411

the mitochonacids

of

and 10% 20%

and

of the is

An-Gal

considerably which

An-Gal

is about

mitochondrial

mitochondrial

and in addition and 17s)

are in

of about

An-Gal+T+E

component

(26s

which

in

values

no detectable

heterodisperse

mitochon-

deficiency

mitochondria

The RNA extracted Id)

cells

the E content

about

15-3076

primitive

90% saturated

for

cyto-

identifiable.

The fatty

with

contam-

four

clearly

cells.

acids

always all

is apparent

compares

RNA isolated

on linear

The resulting

fatty

the

However

grown yeast

In addition

weight.

lb, extent,

are made up of about

and 80% unsaturated

mitochondria

Figure

the most

composition

can

rRNA components

are resolved

from An-Gal

either

species

and are individually

isolated

from

The mitochondrial

rRNA species.

lipid

The presence

(II).

grown yeast

to a variable

and E (1).

membrane

structures

Aer-EtOH

WA's

component

cytoplasmic

in anaerobically

respectively,

anaerobically

and biochemically

to be found

from

21s and 15s

isolated

As shown in is,

gradients,

of both

b.

the

a 4-5s

mitochondria

rRNA species

Morphologically

depleted

RNA species

la,

le.

some cytoplasmic

and mitochondrial

are

transfer

sedimenting

mitochondria

plasmic

dria

with

in

size,

subunits,

together

in Figure

with

include

and smaller

or lipid-supplemented

is illustrated

(26s and 17s)

the larger

RESEARCH COMMUNICATIONS

and molecular These

mitochondrial

well-defined

be compared

composition

ribosome

of these

cells

AND BIOPHYSICAL

with

rRNA

an approximate

small contaminated

amounts the

of

Vol. 43, No. 2, 1971

BIOCHEMICAL

d

10

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

20

30

FRACTION

40

50

NUMBER

Figure 1. Sucrose density gradient centrifugation of mitochondrial An aliquot containing and cytoplasmic RNA from 2. cerevisiae. approximately 30 pg of RNA was layered onto a 5 ml linear gradient of 15 to 30% ribonuclease-free sucrose containing 5mM KCl, 15ti Tris-HCl, pH 7.5 and centrifuged at 32,000 rev/min for 17 hours. The gradient was displaced from the bottom using 70% sucrose and Direction of sedimentation was analysed continuously at 254 nm. from right to left. The S values for the individual RNA species are assigned relative to those obtained for Escherichia coli. (a) (b) (c) (d)

Mitochondrial HNA Mitochondrial RNA RNA from Aer-EtOH Mitochondrial RNA

preparations.

Mitochondrial

content

whereas

cytoplasmic

species

to possess

found

of 285, the

6-14s

heterodisperse cannot

Considering (l2,l3)

that it

rRNA has a G+C (guanine

RNA species

be a simple

is not

likely

a G+C content

product

the

with

of about

the mitochondria

of the mitochondrial

mitochondrial that

+ cytosine)

rRNA is 47% G+C and we have

associated

breakdown yeast

from Aer-EtOH cells from An-Gal+T+E cells cytoplasmic ribosomes from An-Gal cells.

DNA has a G+C content 6-14s

412

RNA species

are

48%.

The

therefore rRNA. of 17-21s the products

BIOCHEMICAL

Vol. 43, No. 2, 1971

of mitochondrial further

transcription;

investigated.

to promote cell;

neither

dria

were 65-70s content

about

components

were 5-10s

DISCUSSION

aerobically anaerobically

chondrial

formed

The addition anaerobic of detectable present

results

ultimate on the depleted

cells There

ations

reported,

investigation. lipid the

depleted mitochondrial

which

is not

mitochondrial

acid

ribosome

composition

formation

initiation the

rRNA synthesis is

directly

are a number

of possible

explanations

may be briefly the mitochondrial receptor

RNA polymerase,

and

dependent

membrane.

normal

413

the

We interpret

and contain

DNA-dependent

or at

by our techniques.

to permit

mitochondrial

has no suitable

growth

of UFA or E to the basic

are viable

may be that

mito-

synthesized

detectable

of the mitochondrial

two of which

grown

when the

anaerobic not

sufficient

that

of cells

of the yeast

limiting

are not

are

grown

However

rRNA synthesis.

to indicate

cells

had an E

cells

and E. structure

by lipid

of a source

medium,

It

acids

rRNA species

mitochondria

molecular

in amounts

mitochondrial lipid

fatty

fatty

from

rRNA is apparently

separately growth

mitochon-

mitochondria the

obtained

of UFA's

is altered

mitochondrial

only

of E and the

whilst

and from

and hence

membrane

conditions,

medium

supplements

composition

RNA sedimentation

and 15s mitochondrial

21s

ethanol with

contain

The An-Gal+T

the An-Gal+E

dry weight

yeast

structures

obtained.

from mitochondria

in

anaerobic

unsaturated.

The unique

isolated

lipid

mg/g

1.2

being

simultaneously

similar

dry weight

whereas

currently

mitochondrial

Id are

mg/g

unsaturated,

of about

readily

0.4

are

rRNA in the

rRNA species,

as shown in Figure

RESEARCH COMMUNICATIONS

and E are required

nor An-Gal+E

mitochondrial

contained

most

UFA's

RNA's

of mitochondrial

An-Gal+T

any detectable

these

Both

synthesis

profiles

AND BIOPHYSICAL

The lipid-

cytoplasmic for

mentioned

ribosomes. the

and are under

membrane binding which

observ-

sites

of the for

has been

Vol. 43, No. 2, 1971

BIOCHEMICAL

shown to be tightly On the

other

bound

a number experiments

ribosome

is

to normal

in

lead

close

for

could

us to suggest

association

be possible

organism, the

of ribosome

synthesis

protein

complete

investigation

ribosome

membrane

composition

in

the

lipid-

cannot

a feedback

inhibition

of ribosome

an ideal

(Forrester

enzymes

membrane

inhibition

changes

and structure

Consequently

are present

mitochondrial

by major

membrane

biosynthetic

system

RNA and ribosome

cells

accompanied

the

and hence This

mutants

mitochondrial

all

and

mitochondrial

(15,16,17).

and constitutes

of the An-Gal

demonstrable,

of it

(14).

genetically

the

mitochondrial

of mitochondrial

aeration

that

formation

occurs.

reversible

both

determined

the

even though

the deformed

incorporate

is readily

with

part

that

rRNA and ribosomal

depleted

described,

membrane

of new cytoplasmically

and may even be an integral it

mitochondrial

hand we have recently

biochemically, and these

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

for

formatior the

synthesis.

rRNA rapidly

On becomes

in the mitochondrial end Linnane,

in

preparation).

REFERENCES 1.

Watson, 88

2. 3.

4. 59 6. 7. 8.

9. IO.

K.,

Haslam,

J.M.

and Linnane,

(1970).

A.W.,

J.

Cell

Biol.

46,

Criddle, R.S. and Schatz, G., Biochemistry 8, 322 (1969). Veitch, B. and Linnane, A.W., in Watson, K., Haslam, J.M., gutonomy and Biogenesis of Mitochondria and Chloroplasts, Eds. Boardman, N.K., Linnane, A.W. and Smillie, R.M. Amsterdam : North Holland, in press (1971). Clark-Walker, G.D. and Linnane, A.W., Biochim. Lamb, A.J., Biophys. Acta 161, 415 (1968). Forrester, I.T., Nagley, P. and Linnane, A.W. FEBS Letters 11, 59 (1970) * Jollow, D., Kellerman, G.M. and Linnane, A.W., J. Cell Biol. z, 221 (1968). Katz, S. and Comb, D.G., J. Biol. Chem. a, 3065 (1963). Rogers, P.J., Preston, B.N., Titchener, E.B. and Linnane, A.W., Biochem. Biophys. Res. Commun. 11, 405 (1967). Wintersberger, E., 2. Physiol. Chem. M, 1701 (1967). Fauman, M., Rabinowitz, M. and Getz, G.S., Biochim. Biophys. Acta 182, 355 (1969). 414

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11.

12. 13. 14. 15. 16. 17.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Morimoto, H., Scragg, A.H., Nekhorocheff, J., Villa, V. and Halvorson, H.O., in Autonomy and Biogenesis of Mitochondria and Chloroplasts, Eds. Boardman, N.K., Linnane, A.W. and Smillie, R.M. Amsterdam : North Holland, in press (1971). Tewari, K.K., Votsch, W., Mahler, H.R. and Mackler, B., J. Mol. Biol. 20, 453 (1966). Bernardi, G., Faures, M., Piperno, G. and Slonimski, P.P., J. Mol. Biol. $3, 23 (1970). Wintersberger, E., Biochem. Biophys. Res. Commun. &I179 (1970). Bunn, C., Mitchell, C.H., Lukins, H.B. and Linnane, A.W., Proc. Natl. Acad. Sci. U.S. a, 1233 (1970). Linnane, A.W. and Haslam, J.M., in Current Topics in Cellular Regulation, Vol. 2 Ed. by Horecker, B.L. and Stadtman, E.R. New York : Academic Press, p.101 (1970). Dixon, H., Kellerman, G.M., Mitchell, C.H., Towers, N.H. and Linnane, A.W., Biochem. Biophys. Res. Commun. in press.

415