Deoxyribonucleic acid associated with yeast mitochondria

Vol.

15, No.

BIOCHEMICAL

2, 1964

DEOXYRIBONUCLEIC

ACID

G. Institut

January

Received

DNA

ASSOCIATED

SCHATZ, E. fur Biochemie,

considered

found

nuclear

BIOPHYSICAL

RESEARCH

WITH

HASLBRUNNER University

YEAST

and H. of Vienna,

COMMUNICATIONS

MITOCHONDRIA TUPPY Austria.

27, 1964

is generally

sometimes

AND

material

to be confined

in other

cell

(Allfrey

1959).

DNA

occurring

in the

somes

(Clark

and

1963)

is well

documented

cause

in these

DNA

nucleus.

usually

presence

of the

1960), but

are

The

cytoplasm

Wallace

cases

fractions

to the

attributed

of significant

amphibian

and

in the

is regarded

chloroplasts

with

amounts

of DNA

to contamination

by

amounts

&cyte

rather

is associated

Small

(Stich

1962),

the

of plant

cells

(Chun

as an exception

cellular

of extranuclear

than

components

kinetoet al.

as a rule,

absent

from

behigher

cells. This

paper

yeast,

purified

DNA.

The

presents

evidence

by flotation

amount

ribonucleotide

found

that

in density is far

component

preparations

gradients,

in excess

(Appleby

contain

of that

and

of mitochondria a significant

accounted

Morton,

from

1960)

for

by

baker’s

quantity the

of

polydeoxy-

of mitochondrial

cytochrome

b2. Since

the

those

of mammals,

cluding the

possibility,

mitochondrion

that

nature

the

is unknown.

factors The

of DNA

DNA

function

genetic

an organelle

occurrence

extranuclear

Mitochondrial chromosomal

represents

in the (Ephrussi

presence

is much cell

and

Hottinguer

of DNA

in yeast

more

yeast

present

in yeast

1951), mitochondria

cells,

mitochondria

common

is in part

in most

that

in-

suggests

hithertosuspected

controlled whose

by extraexact

is thus

chemical of special

interest.

Experimental (Schatz

: The wild type strain W of Saccharomyces

1963).

The concentration

yeast homogenates et al.

1963).

contained tion,

except

of glucose

and of the crude mitochondrial that the medium

0.25 M mannitol,

a linear continuous

employed

20 mM tris-buffer sucrose gradient

cerevisiae

in the growth

(volume

fractions

medium

was grown as described

was the same as in earlier

for the homogenization pH 7.4 and 1 mM EDTA. 25 ml: 0.98-l.

127

previously

was 0.8 %. The preparation

and washing For density

work

of the (Schatz

procedures gradient

66 M sucrose; additions

flota-

as above)

Vol.

15, No.

2, 1964

was constructed fraction

BIOCHEMICAL

AND

ultracentrifuge.

If sedimentation

involving

through

triiodo-benzoate.

flotation

an X-ray

the gradient

follows

: The crude mitochondrial

ficient

“Urografin”

fraction

as described

above.

After

from the top of the gradient

diluted

with homogenization

and an aliquot

activity

and acid-soluble

extracted

with 0.5 M perchloric followed

obtained

with this procedure

Button (1956). with pancreatic containing

within

DNase (Fluka)

150 pg/ml

pure cytochrome protein

pressed as pmoles : An

examined.

and

the

This

lack

the

removal

enzymic

of information

purification

ally

with

high

density

ever,

it was

come

these

initially

+) We are greatly

density Thus,

the preparation

indebted

c reductase

from as the

nucleus

and

by enzymatic

gradient

examined equilibrium

equilibrium of pure

thus

far

yeast

to Dr. Klose (Fa. Schering.

substance. 128

Vienna)

fracproperties

assessment

In view

not

known.

(Allfrey

for of their

of the

exception-

1959),

how-

might

in sucrose

mitochondria.

the

are

centrifugation

centrifugation

of 3.0 ml at 20’.

centrifugation

as the

tests.

are ex-

sedimentation

of differential

of

and that of

activities

mitochondrial

its fragments

as well

was

number

requires

the

pH 6.6

activity

activity

DNA

fragments

buffer

Specific

of mitochondrial

fractions

nuclei

1964).

and

Digestion

of the turnover

in a total volume

adoption

The results

of Dische (1930)

c reductase

for measurements

problems,

the

the DNA

1952).

per mg protein

yeast

material

isolated

that

difficulties. for

difficult

mitochondrial

of all

(Ceriotti

was

lipid was also

Finally,

error was always observed.

and Klima

nuclear

of the

nuclear

hoped,

tried

poses

precluded

of the

contamination

and

complement

In some experiments

L-lactate-cytochrome

(Schatz

demonstration

task

of the particles

by use of the methods

(1961)

at 30 000

c reductase

out at 26’ for 2 hours in 0.02 M acetate

pet minute

and

in homogenization

The remainder

at 0’.

suf-

They were then

succinate-cytochrome

of succinate-cytochrome

of nuclei

This

were resuspended

experimental

elsewhere

reduced

unambiguous

quantitative tions

of acceptor

syringe.

3 : 1 at room temperature.

and 0.05 M MgCl2.

as described

1.20 g. cme3)

were washed twice by centrifuging

checked of

containing

of 3.0 ml each were collected

designed

by the indole method

chosen by Nygaard

b2. The determination

were performed

Results

rhe limits

medium

4.5 ml of this heavy

(1.10

fractions

acid in 35 70 ethanol

was carried

enzyme

assayed under the conditions

gradient

compounds,

were frequently

Agreement

were conducted

to 1.25 g. cmu3.

The pellets

was determined

as above.

as

for the assay of protein.

-ether

and centrifuged

by Fa. Schering)?

centrifugation,

by ethanol

of these washed particles

gradient

6-

” Urografin’

deoxyribose-containing

4.5 ml of a mitochondrial

in homogenization

and the particles

was withdrawn

with ethanol,

content

gradient

medium

of a crude mitochondrial

(N, N’-diacetyl-3,5-diamino-2.4,

with the help of a specially

in the Spinco no. 30 rotor.

medium

washed twice

was dispersed of the suspension

by 25 ml of a linear

starting

rpm for 60 minutes

of * Urografin”

agent manufactured

to adjust the density

suspension were overlayered

was desired.

on top of the preformed

in gradients

contrasting

COMMUNICATIONS

at 25 000 rpm for 5 hours in the IOIOI SW 25.1 of the Spinco

SusPeosion in 0.5 M suctose were layered

centrifuged

RESEARCH

(Bock and Ling 1954) on top of 4.5 ml of a suspension

in 1.8 M sucrose and centrifuged

Experiments

BIOPHYSICAL

The

for the generous

over-

gradients resolution

supply with this

was of

BIOCHEMICAL

Vol. 15, No. 2, 1964

subcellular

components

satisfactory

for

the

a well-defined within

mitochondria

were

was

nuclear

fragments

gradients,

other

“Urografin”

types

obtained

by floating

schematically

depicted

which

also

crude

mitochondrial

of this

appeared

experiments,

protein

(table two

to DNA

well

content

widely

peaks

with

particles

protein.

and

to DNA

other

tions and drial

other

compounds (table

is not Morton

I).

not The

accounted 1960)

concentration

for

of mitochondrial of cytochrome

detergent

c reductase activation.

(The

analyzed

of the the

the

was due

mitochondria.

nuclear

matter)

from

20 - 250&mg

with

the

peak

The varied of

of mitochondrial

digestion

with

pancreatic

proved

to be due DNase,

rendered

acid-soluble.

In addition

3.5-4.8

rJg/mg

of deoxyribose-

by acid, found

in the

cytochrome

b2.

in the

of activation

129

mono-

purified

Table

mitochondrial

from

II gives the

purified by the

frac-

(Appleby the

specific mitochondrial detergent

to

or oligonu-

component

b2 as calculated measured

protein

presumably,

polydeoxyribonucleotide

extent

for

experimental

Upon

of DNA

mg of

peaks

thus

precipitable

by the

were

and

contained

particles

per

activity

was

of the

DNA

under

B,

mitochondria.

c reductase

DNA

amount

the

from

exactly

in band

microscope,

One

bands

are

activity

floated

ranged

The

c reductase

including

and

trials,

was found

4.3~~19

3).

not

sedimenting

of gradient

gradient

separated

coincided

type

the

work.

of yeast

and

the

of sucrose

present

protein

(figure

(probably

mitochondria.

mitochondria

L-lactate-cytochrome after

of DNA

mitochondrial

the

electron

the

through

numerous

in this

of the

were

clearly

material to the

with

purified

containing cleotides

thus

contained

shortcomings

for

1.1

fractions

succinate-cytochrome

associated

the

dense

peak

up to 75 % of this DNA,

were

oneexperiment

The

protein

and

between

which

large

to slowly

population

found

relatively

Essentially

After

of the

in the

were

between

which

due

suited

homogeneous

different

separated

of this

from

the

separation

mitochondrial.

the

formed

to be diffusely

of sedimentation)

bulk

If viewed

found

band

succinate-cytochrome

contained

When

employed

The

to be un-

mitochondria

The

in the

fractions

4.

as a fairly

associated

conditions DNA

I).

in DNA.

investigated.

to be well

of the

they

no clear

to avoid

mitochondrial

most

was

complications

were

found

fraction.

In five

DNA,

In an attempt

in figure

contained

band

2).

crude

was

(instead

to avoid

were

DNA

as truly

in order

of gradients

gradients

-3,

found

if flotation

(figure

the

rich

be regarded

proved

Whereas

l).There

of protein)

obtained,

employed

work.

particles

not

however,

I. 18 g.cm

gradient(figure

thus

procedure,

present of

(20-4Oug/mg

could

gradient

of the

contaminating

of DNA

results

by this

at a density

the

and

mitochondria same

purpose

band

distributed

amounts

effected

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mitochonactivity

of fraction

as we,lI

as

Vol. 15, No. 2, 1964

BIOCHEMICAL

AND BIOPHYSICAL

‘B 2.0 -

-10 2.0 -

1.13

1.16

l.lq 1-b

1.13

RESEARCH COMMUNICATIONS

c

%j.3

““.. .

1.16

1.1-l I.22

1.10

143

Cl6

I.19

Figures 1 ~3 : Distribution of succinate-cytochrome c reductase activity (. . . . ), protein (- -- - -) and DNA (. -. -. -) after subjecting crude preparations of yeast mitochondtia to sedimentation in a sucrose gradient (fig. 1). flotation in a sucrose gradient (fig-S) and flotation in a “Urografin” gradient (fig. 3). Abscissa : density (g. cms3). Ordinate A : mg protein/ml fraction and A E550 pet minute per 0.4 ml fraction: ordinate B : fig DNA/O. 1 ml fraction: ordinate C : pg DNA/ml fraction.

A 6

Figure 4 : Banding of a crude mitochondrial fraction from yeast in a ” Urogtafin” gradient. A (1.12 g. cme3) : Mitochondtial precursors (Schatz 1963) B (1.15 g. cmm3) : Mitochondria C (1.19 g.cme3) : Double Membranes rich in ATPase (Schatz et al. 1963) D () 1.25 g. cmm3) : Debris including nuclear material. -

c

H

0

Fig.4 Deoxyribose compounds not precipitable by acid ++I ,ug / mg protein Experiment Experiment Experiment Experiment Experiment

Table

1 2 3 4 4a 5 5a

3.48 4.25 4.83

Deoxyribose compounds precipitable by acid (DNA) ,ug / mg protein 1. 55 1.18 1.06 1.51 3.60 4.30 4.30

5) +) &) &) f)

I : Deoxytibose compounds in purified yeast mirochondria. ++) Calculated as nucleotides f ) Particles incubated at 26O for 30 min at pH 6.6 after acid precipitation + ) Particles incubated at 26’ for 2 hours at pH 6.6 after acid precipitation & ) Lipid extracted as described under Experimental.

1.30

BIOCHEMICAL

Vol. 15, No. 2, 1964

Enzyme

tested

Specific without

L-lactatecytochrome reductase

c

succinatecytochrome reductase

c

Table

the

activity

detergent

activity

0.054

0.12

0.39

nil

of succinate-cytochrome

into

cleotide

account,

(Appleby

DNA

found

the

of the

pellet

found

in the

Discussion

for

2 hours

of DNA

from

supernatants

purified

mitochondrial

that

under

wos

assembly yeast

are

and

The the

chemical

the

1963).

information

mitochondrial

mitochondria amounts

of nucleic

the function

themselves. of DNA

flotation.

The the

This

associated material

deoxyribose

acid of the

possibility

was

in the

the

to arise

The the

and

but

as the that

primary

these

paper

reports

of small,

content

was (assayed

131

identified with

available

1955;

DNA

in question

and

Simpson

detection

in

(Ephrussi

is unknown,

of DNA

mitochondria

present

of mitochondria factors

from

organized

be extrochromosomal

yeast

DNA

previous

might

with

rotor).

the

appear

investigated

the

parti-

no.40

1963).

(Rout role

10 to 40,

that

by

function

factors

I n view

found

controlling

genetic

of

contaminant.

Schatz

and

of these

of

unlikely

mitochondria

factors

amounts

(Spinco

prompted

1962;

Structure

nature

involvement

Marcovich

of genetic

precipitability,

about

by extrachromosomol

governed

suggests

gradient

more

et al,

in part 1951).

regulating

ficant

to learn

it very

yeast

structure.

Moustocchi carrier

(Linnane

rpm

was an adsorbed

conditions

the

b2. was

at 40 000

were

comparison).

by a factor

homogenates

here

of mitochondrial

Hottinguer evidence

certain

that

cytochrome

made

described

precursors

undertaken

be calculated,

fractions

investigations

for

c

% of polvdeoxyribonu-

exceed,

centrifugation the

included

5-6

by mitochondrial

present

and of succinate-cytochrome

are

preparations

yeast

non-mitochondrial work

it con

in the

: The

observations,

c reductase

b2 contains

1960)

contributed

upon

absence

cytochrome

Morton

DNA

0.11

c reductase

mitochondrial

of DNA

All culate

and

in our

amounts

The

that

Calculated concentration of cytochrome b2 (% of mitochondrial protein)

with 0.1 % Triton X 100

II : Specific activities of L-lactate-cytochrome reductase in purified yeast mitochondria.

Taking

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

factors

present

within

but

signi-

purified

by density

as DNA

by its ocid-

different

methods)

and

its

Vol.

15, No.

2, 1964

digestion

BIOCHEMICAL

by pancreatic

mitochondrial

fraction

excluded,

since

from

the

drial

marker

bulk

regarded

results

of the

obtained

presence pounds

that

of DNA

here.

The not

the

might

of appreciable

amounts

phenomenon.

Acknowledgements Public Health the preparation

This

this

that

the

the

occurrence

have

been

found’in

in the

of small

amounts

possibility

is being

: This work was Service. The authors of the manuscript.

of the

whole

band

well

peak

of the

other

cells

ratio

possibility from cannot

virtually

to the

controlling

Moreover,

the

mitochondria of extranuclear

com-

metabolism all

of yeast DNA

plant may

the

support

deoxyribose-containing of DNA

the

to corroborate

factors DNA.

to be

of mitochon-

lends

extrachromosomal

be

mitochon-

but

in order

in mitochondria

can

of DNA

base

the

separated

unlikely,

the

with

material

absence

be mitochondrial

suggests

associated

The

comparing

of acid-soluble,

of DNA

only

is considered

of DNA

in yeast

occurrence

Because

from

function

the

c reductase.

extracted detection

DNA

COMMUNICATIONS

by nuclear

with

at present

it,

detection

the

in a discrete

coincided

homogenates

We are

Mitochondria The

to occur

artefacts.

that

in mitochondria

cells.

found

proving

organelles.

that

RESEARCH

to contamination

exactly

yeast

out.

though

mitochondrial

possibility

be due

of adsorption

with

BIOPHYSICAL

succinate-cytochrome

ruled

hypothesis,

The

was

and

enzyme,

portion

DNA

DNA

of DNA

be definitely drial

might

this

is that

soluble

DNase.

AND

in these and

thus

is a rather

animal indicate, general

investigoted.

supported by grant GM 11225-01 are indebted to Dr.R.Wolfe for

of the his help

Allfrey,V.,

in : “The Cell” (Brachet, J. and Mirsky,A., ed.). Academic 1959, vol. I, p. 193. Appleby, C.A. and Morton,R. K., Biochem. J. 75, 258 (1960). Bock,R.M. and Ling.N.S., Analyt.Chem.26, 1543 (1954). Burton,K., Biochem .J. 62, 315 (1956). Ceriotti,G., J .Biol. Chem. 198, 297 (1952). Chun,E.H.L., Vaughan,M.H. and Rich.A., J.Mol.Biol.7, 130(1963). Clark,T.B. and Wallace,F.G., J.Protozool. _7, 115 (196@. Dische,Z., Z.Mikrochem. 2, 26 (1930). Ephrussi, B. and Hottinguer, H., Cold String Harbor Sympos.Quant.Biol.

U.S. in

Press

3,

(1951). Linnane,A.W., Vitols,E. and Nowland,P.G., J.Cell Biol. 2, 345 (1962). Moustacchi,E. and Marcovich,H., Compt. rend. 256, 5646 (1963). Enzymes” (Falk,J.c Lemberg.R. and Morton,R.K., Nygaard,A.P., in : “Hematin ed .) Pergamon Press 1961, p. 547. Raut,C.W. and Simpson,W.C., Arch.Biochem.Biophys. 57, 218 (1955). Schatz,G, , Biochem. Biophys.Res. Communic. 2, 448 (1%3). Schatz,G., Tuppy,H. and Klima, J., Z. Naturforsch. g, 145 (1963). Schatz,G. and Klima,J., Biochim.Biophys.Acta, in press. Stich,H.F., Exp.Cell Res. 2, 136 (1962).

132

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