Vol. 70, No. 2,1976

BIOCHEMICAL

EFFECTS

OF TEMPERATURE

INTERACTIONS

IN

N. MARMIROLI, 43100 February

THE

RESEARCH COMMUNICATIONS

ON NUCLEO-MITOCHONDRIAL

YEAST

F. RESTIVO,

Institute

Received

AND BIOPHYSICAL

SACCHAROMYCES CEREVISIAE

E. ZENNARO, P.P.

of Genetics,

University

PUGLISI

of Parma,

PARMA, Italy

27,1976

Summary: The temperature, as well as several antibacterial antibiotics could be used to differentiate mitochondrial protein synthesis (MPS) from the cytoplasmic (CPS) one in the yeast Saccharomyces cerevisiae. In fact MPS and CPS have respectively the optimum at 30°C and 36 0C. A series of cellular processes,,.as the mitotic reproduction in presence of non-fermentable carbon sources, the synthesis of galactose pathway enzymes and the meiotic process have the same optimal temperature (30OC),whereas the growth of the wild type in presence of fermentable carbon sources and of a galactose repressor constitutive mutant (i-) have the optimal temperature of 36'C, in agreement with our previous hypothesis in which the expression of some sections of the nuclear genetic complement is dependent on regulatory functions controlled by MPS.

INTRODUCTION It

is

well

tem for (CPS)

established

protein

that

synthesis

in

(MI'S)

the yeast differs

in

Saccharomyces several

cerevisiae

aspects

from

mitochondrial the

cytoplasmic

sysone

(I,2,3).

We have

therefore

functionsas

well

indicate

to depend

Our results is 36°C

analyzed as of

on the

show that

in vivo

some biological activity the

the

dependence

processes

on the

which

other

temperature kinds

of

the

of evidence

above seem

of MPS (4,5).

optimal

temperature

for

MPS in vivo

is

30°C and for

CPS

.

MATERIAL AND METHODS Yeast

strains

. S. cerevisiae

ilv I, i- (galactose Culture Media.

The following

-Complete

M (6) with

medium:

5300 mat I-A/B

prototroph,

and 535217d

mat I-A,

ura3,

constitutive). culture glucose

(V/V) or galactose 2% (W/V) as carbon - Sporulation medium: 23DHA (6).

media 0,6%

have

(W/V),

been used: or glycerol

2% (W/V)

or ethanol

2%

sources.

For the description of the strains we have used the nomenclature indicated by M.E. Plischke, R.C. von Borstel, R.K. Mortimer and W.?,. Cohn in "Genetic markers and associated gene products in Saccharomyces cerevisiae.

Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

589

BIOCHEMICAL

Vol. 70, No. 2, 1976

04 24

26

AND BIOPHYSICAL

28

30

32

Temperature

a.

34

36

RESEARCH COMMUNICATIONS

38

40

I”C J

b.

Fig.

strain 5300 in presence la - Optimal temperature for the growth of S. cerevisiae of different carbon sources. (*!m; (-O-+-) galactose; (w) glycerol; (*) lactate; ) ethanol. Growth rate A = To/Tx is indicated as the ratio between the division time at the optimal temperature (To) and the division time at the different temperatures indicated (TX).

Fig.

lb - Optimal temperature for the growth (IlD->, S. cerevisiae 5352/7d; galactose constitutive (i-). Division

in the

time.

complete

The samples, the with tures

beginning the for

The cultures were grown overnight medium with 0,2% glucose as carbon collected

of

with (a-))

the

by centrifugation

stationary

carbon

sources

the

determination

phase,

indicated of the

(3500xg were

in Fig.

galactose as carbon S. cerevisiae

at 28°C on alternating source. for

resuspended

10'at

time.

590

at

medium the

:

shaker

room temperature)

in fresh

la and lb and kept

division

source of 5352/7d;

at

supplemented

scheduled

tempera-

Vol. 70, No. 2, 1976

Fig.

BIOCHEMICAL

2 - Optimal

temperature

Sporulation.

of the - cells concentration of 4.10'

final 10'

at room

temperature),

sporulation

medium

for

strain

as previously

ferent

temperatures

(specific proteins mczky

was determined (7)

at

the

filter

synthesis indicated

- Diphenyloxazole

the

radioactivity

to a for

cells/ml

indicated

in the in Fig.

0.5Ni/ml

and ex-

in vivo.

The cells,

protein

synthesis

at dif-

of

l-(14C)

aminoacids

incorporated

tecnique

2.

synthesis

cytoplasmic material

was estimated 3a,

materials,

overnight

was dissolved

2.5

protein

by adding

in Fig.

radioactive

by keeping

2.10'

until (3500xg

of sporulation was estimated at the optimal temperature.

for

filter

of

temperatures

5300.

in

as decribed

mixture

the

cytoplasmic

by Bilinski

and Jachy-

(8).

protein

temperature

solubilized

3a,b)

described

concentration

The radioactive

by Millipore

and by Kennel

The mitochondrial Each

Fig.

assayed

strain

by centrifugation

and mitochondrial

52mCi/mA).

Mitochondrial (6)

(see

as previously

different

on the degree sporulated,lcells

were

RESEARCH COMMUNICATIONS

of S. cerevisiae

collected the

at

at the

described,

activity

grown

were

resuspended

and incubated

of cytoplasmic

grown

sporulation

5300,

cells/ml

The effect of the temperature pressed as the percentage of the Determination

AND BIOPHYSICAL

the in

as described

samples

of p-bis

was determined

and Kaudewitz

b. adsorbed

to Millipore

in 1 ml of

10 ml of Toluene

and 0.05%

by Schweyen

based

soluene

mixture

- Benzene)

spectrometer

(0.25)5),

were

350R.

scintillating

(0-Methylstryryl

in a Beckman

filters

(0,5%

in Toluene

of

and

LS 100.

RESULTS AND DISCUSSION I - Optimal

temperature

As shown in Fig.3a nificantly CPS: the

ME'S and CPS in vivo.

and 3b the

optimal

temperature

for

MPS and CPS in vivo

are

sig-

different. incorporation

shows a two-fold 36°C

for

(optimal

of

14 C - labeled

increase temperature)

between

aminoacids 25"

and 3O"C,

and decreases

591

in Erythromycin achieving

at negligible

inhibited its

value

maximal at 45°C.

cells value

at

BIOCHEMICAL

Vol. 70, No. 2, 1976

AND BIOPHYSICAL

,

1

22

26

30

34

Temperature

Fig.

RESEARCH COMMUNICATIONS

3a - Optimal temperature S. cerevisiae strain

38

42

46

(Oc 1

for CPS (m) 5300

and MPS (d--o-)

rI

I

1100 -

900

600

in the

diploid

7 P vh

-

700

500

400

300

-

200

4.=; i 2”

300

-

J 22

Fig.

3b - Optimal revisiae

temperature for strain 5352/7d.

MPS: the incorporation tion increasesrapidly negligible

value

2.5

30

34

Temperolure

b.

of

CPS (m->

38

42

100

3 ?

46

IT!

and MPS (-04)

inhaploid

14 C - labeled aminoacids in the Erythromycin sensitive from 20°C to 30°C (optimal temperature), decreasing

at 45°C.

592

-.S. ce-

fra. at

Vol. 70, No. 2, 1976

Therefore,

the

MPS from

optimal

temperature

CPS in vivo,

thromycin,

for

carbon

As shown in Fig. with

glucose

the

growth

with

the

as sole

that

differentiates

to some antibiotics

such as Ery-

(10,11,12). of

S. cerevisiae

is

temperature

temperature

carbon

source

is

for

the mitotic

36"C,

whereas

reproduction the

of

optimal

S. cerevi-

temperature

for

difference

in

30°C.

and galactose

the optimal

parameter

sensitivity

reproduction

optimal

galactose

glucose

RESEARCH COMMUNICATIONS

sources

la

siae

to the

and Tetracyclines

temperature

a) Fermentable

AND BIOPHYSICAL

seems to be another

in addition

Cloramphenicol

2 - Optimal

Since

BIOCHEMICAL

are both

could

fermentable

be explained

carbon

sources,

on the basis

this

of one of the

following

hy-

pothesis: I-

The optimal

to the

temperature

Leloir

pathway

2- The optimal of

the

case

inducible

for

galactose coding

of

for

for

mutant

the

As shown

repressor

36"C,

coincident

This

observation

lb,

coincides

with

galactose

as carbon

The fact as carbon

the

that source

that

tion

and is

inducible mum lies in the

the

at 3Q"C,

optimal

for

glucose

its

expression,

(13,14),

is 30°C.

the

adaptation

carries

not

be-

as in the

and growth

a mutation

with

in the

gene

the

growth

of

the

constitutive

strain

utilization. 1) and 3) and agree

temperature

optimal

with

the hypothesis

2).

temperature

for‘the

lactate for

growth

and ethanol,

the

growth

in presence is

the

of non-

same (3O'C)

of an inducible

strain

and

with

source.

the

galactose

in respiratory has the

induction

repressor

with

system,

the hypothesis

as glycerol,

sensitive strain

&

for

sources

of MPS as Erythromycin tion

for

is 3O'C.

sources

the

carbon

system

function

of a function

for

temperature

temperature

optimal

discharges

in Fig.la,

fermentable

optimal

the

carbon

for

enzymes"

activity

needed

sensitive

belonging

of a regulative

"galactose

is

enzymes

3O'C.

activity

or the

presence

the

(5).

the

with

b) Non fermentable As shown

(i-)

is

of the

optimal

of one of

or the

synthesis

repression

constitutive

?n Fig.

catabolism

synthesis

whose

the

in vivo

synthesis

the

but

determined

i3

the

the

temperature

activity

galactose

for

system

then

the

the

to the -gal system of CPR for catabolite

We have

is

for

temperature

3- The optimal longing

for

of to

grows

at

whereas (15)

constitutive

(i-)

deficient

condition

optimal

galactose the

above

growth system

the

make the

constitutive regulatory

with

is blocked (5),

as enzyme

593

at 36"C,

with

induction

as well

grow

indipendent

at 36"C, from

the

deficient

be explained dependes

galactose

of an inhibitor

in respiratory

could

one could system

grows

and in presence

temperature

antibiotic

3O'C insofar

strain,

assuming

observa condithat

on MPS, whose since the

the opti-

the mutation

activity

of MPS.

BIOCHEMICAL

Vol. 70, No. 2, 1976

Sporulation IA/B

. In a previous

yeast

to

tromycin, controlled

strain

then

2, is

determined 3O"C,

utilized

the

the

with

hypothesis

(5)

the

deficient

commitment

protein

coincides

fact

with

system

coincident process,

the

coincides gal

attributed

respiratory

at

the optimal

which

that

in an inducible

diploids

we have

failure

(4).

temperature

for

the

of heterozygous

conditions

optimal

mat

or in presence

to sporulation

synthesis

with

RESEARCH COMMUNICATIONS

on regulatory

sporulation,

which,

temperatures

for

of Ery-

functions as shown

MPS in the

diploid

(Fig.3a).

In conclusion galactose of

in the

dependence

by the mitochondrial

We have in fig.

sporulate

to the

paper

AND BIOPHYSICAL

the

optimal

in which

are mediated

the

optimal the

temperature

temperature induction

for

for

sporulation for

temperature

by regulatory protein

temperature

and ii)

optimal

has an optimal the

on the mitochondrial

the

strain

for

the

CPS, is

of

(i)

the

in the

MPS, whereas growth

of the mat IA/B

a constitutive

in presence

in agreement

the -gal system, factors whose activity

utilization

heterozygous

as well and/or

with as the

mutant

of galactose our previous sporulation

synthesis

depends

synthesis.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Barnett, W.E. and Brown, D.H. (1967) Proc. Nat. Acad. Sci., 11, 425, Barnett, W.E. and Brown, D.H. (1967) Proc. Nat. Acad. Sci., 57, 1775. Kuntzel, H. and Noll, H., (1967) Nature, 215, 1340. Puglisi, P.P. and Zennaro, E., (1971) Experientia, 27, 963. Puglisi P.P. and Algeri, A.A., (1971) Mol. gen. Genet. 110, 110. Magni, G.E. and van Borstel, R.C., (1962) Genetics, 47, 1097. Bilinski, T. and Jackymczyk, W., (1973) Biochem. Biophys. Res. Commun., 52, 379. Kennel, D., (1967) Methods in Enzymology, XII, 680, Academic Press, New York. Schweyen, R. and Kaudewitz, F., (1970) Biochem. Biophys. Res. Commun., 38,728. Clark-Walker, G.D. and Linnane, A.W., (1967), J. Cell Biol., 34,l. Lamb, A.J., Clark-Walker G.D. and Linnane, A.M. (1968) Bioch.?iophys. Acta, 161, 415. Linnane, A.W., Lamb Z.I., Christodoulou, C. and Lukins, H.B., (1969). Proc. Nat. Acad. S&., 2, 1288. 13. sy, J. and Richter, D., (1972) Biochem., 11, 2788. 14. Sy, J. and Richter, D., (1972) Biochem., 2, 2784. 15. Puglisi, P.P. and Algeri, A.A. (1974) Ateneo Parmense, act. nat., lo, 455.

594