Nuclear and cytoplasmic glutamate dehydrogenases (NADP-dependent) in Saccharomyces cerevisiae

BIOCHEMICAL

Vol. 64, No. 2, 1975

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

NUCLEAR AND CYTOPLASMIC GLUTAMATE DEHYDROGENASES (NADP-DEPENDENT) IN SACCHAROMYCES CEREVISIAE Laura

Camardella,

Guido

International

Institute Marconi

Via

Received

April

4,

di Prisco,

Felice

Garofano

and Anna Maria

of Genetics and Biophysics, 10, 80125 Naples, Italy

Guerrini

CNR,

1975 SUMMARY

The intracellular localization of NADP-dependent glutamate dehydrogehase has been studied in Saccharomyces cerevisiae. has been found to be associated Beside cytoplasmic GDH, enzyme activity with the nuclear fraction in amounts comparable to those reported in nuclei of higher organisms. The yield and distribution of both GDH aCtivities have been analyzed in mutants showing, under particular growth conditions, defective mitochondrial functions. INTRODUCTION NADP-dependent has been described in higher

GDH, in lesser

organisms

this

GDH of yeast

tions

(5,6).

bound

to mitochondria, Several

investigation also

yeast

it

nuclei

that

investigated

mic and nuclear

enzymes

tions

a relatively

of the cells,

nuclear

using

differentiate the possibility

is

regulated mutants

local-

cytoplasmic

frac-

GDH associated

(7-13).

reported

below

GDH level. it

from

demonstrate

the

and

(14):

cytoplasmic

the synthesis

it

enzyme.

of the cytoplas-

the physiological cerevisiae

the

that

The purification elsewhere

in dependerceof of Saccharomyces

with

We have extended

be reported that

weakly

localization.

another

tissue

high

Although

the enzyme may be very

found

GDH will

the presence (4).

of mitochondrial

soluble

have a different

from animal

clearly

We have

mitochondrial

organism

and the experiments

of yeast

shows properties

in the

cerevisiae

(1,2,3);

been reported

believed

found

have recently

fraction contain

characterization

latter

may indeed

investigators to yeast,

been

Saccharomyces

authors

has also

enzyme was commonly in the

nuclear

of yeast

by several amounts,

has often

Although

the purified

dehydrogenase

and characterized

of NAD-dependent ization,

glutamate

defective

condiin

functions. EXPERIMENTAL

Strains: The following yeast strains of Saccharomyces 1511 (15) and an isogenic derivative -pts mutant strain Growth conditions: Cultures were grown in YEP medium

Copyright All rights

o I9 75 by Academic Press. Im. in any form reserved.

of reproduction

773

cerevisiae were rho- 1511-1. consisting

used:

of 1% Difco

the

Vol. 64, No. 2, 1975

yeast extract the medium.

BIOCHEMICAL

and 2% Difco Cultures were

Respiration polarographically

peptone; grown

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1% glucose and 3Z 23°C and 36°C with

at

The oxygen uptake a KM Gilson Oxigraph.

measurements: with

of

the

glycerol vigorous

whole

were added shaking.

cells

was

to

measured

Preparation of protoplasts: Early stationary phase cells were treated according to the procedure of Kov~? (16) for preparation of yeast mitochondria, except that snail gut juice was substituted by Glusulase. When protoplasts were formed in 80% yield, they were disrupted in a Sorvall Omni Mixer for 30 sec. at the maximum speed without previously washing away the Glusulase. In fact, it was observed that GDH was partially extracted from the protoplasts by the lytic enzyme, which however did not interfere with the GDH assay. The enzyme activity of the homogenate was assayed Enzyme assay: metrically at 30°C, in cuvettes of l-cm light path, by recording in absorbance at 340 nm of the reduced coenzyme. In a final volume the mixture contained: 0.1 M (NH&SO4, 0.1 M phosphate buffer pH a-ketoglutarate and 0.1 mM NADPH. Activity is expressed as pmoles oxidized per minute, using a molar extinction coefficient of 6.22 one I.rmole representing one activity unit. Reagents: Endo Lab., the other

Erythromycin used as lactobionate NADPH from Calbiochem, a-ketoglutarate reagents were of the highest purity RESULTS

Nuclear

gene

mitochondrial able

substrates

obtained

by

growing

rise

The

the

as

cytoplasmic

pathway. in

we

the

have

mutant

mutations

However,

cells

The

presence

used

can

same

of

yeast

respiratory-deficient

pts

from Abbott, Glusulase from Pierce Chemicals. commercially available.

(18,19).

cells

below,

and

36”C,

well

yeast

was

from All

AND DISCUSSION

glycolytic

yeast

to

1511

structurally at

the

reported

give

as in

using

experiments which

mutations

functions

spectrophotothe decrease of 1 ml, 7.0, 0.013 M of cofactor X 103 (17),

can

affect

the

grow

on

ferment-

phenotype

can

erythromycin

strains

be

(20).

and

growth

For

the

conditions

phenotypes.

strain,

functionally

grown

at

unaffected

mitochondriaare

23°C

and

used

as

mitochondria.

altered

and

the

control,

After

“petite”

develops

prolonged

phenotype

growth

is

expressed

at

23°C

(15,21). The 36°C

for

800

X g,

lo-15% and

about

a few

20 of

by

bound results

of of

cells

in

was the

The 0.32

could

at

36’C

in

are was

the

the

shown twice

the

by in

Table

that

of

774

homogenate the nuclei,

pellet,

supernatant

of

(221,

the

ultrasonic 1. the

even was

purification

2 M sucrose

extracted

the

contained in

further

grown

supernatant;

which

until

in be

cells of

remained

After

experiments grown

found

activity

centrifugation

of

centrifugation

precipitate

M sucrose,

and

homogenates

After

activity.

nuclei these

in

activity

cells.

high-speed the

measured

with

enzymatic

to

the

associated intact

of

was generations.

washing

devoid

gram

level

85-90%

was

repeated

tion

GDH

The

activity

yield at

at

remaining cell

debris

after

completely the

treatment

control

and

nuclear was

fracstill

(14). of

23OC,

enzyme but

The per the

BIOCHEMICAL

Vol. 64, No. 2, 1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Distribution

of

GDH activity grown at

23°C

fraction

1 in

23°C

(1.3

(1.8

g cells)

units/g

100

89.7

1.116

89.8

800 x g sediment

0.054

10.3

0.126

10.2

of

for

activity the

and

grown

completely

at

(21).

in

amount

these

The the

enzyme

cultures

of

GDH

of per

the

the

and

1511

strain

of

cells

cultures

of and

the

was

1511

of

mutant

36’C:

functions,

two-fold

in

as

the

terms

in

these

than

that

are

distribu-

comparable

higher

is

content,

well

also

mg/

strain

cytochrome

homogenate,as

at

2.5

phenotype

mitochondrial and

strain,

absence

rho-

fractions,were

grown

the

constant.

respiratory-deficient

the

cytoplasmic

was

presence

synthesis in

fraction

spontaneous

The

content and

gram

in

of

protein

nuclear

nuclear with

23°C

cases

mitochondrial

between

the

temperature.

in

1.240

obtained at

cultures

same

expressed

found

results

with

the

respiration,

blocked

with

20 generations

erythromycin,

tion

associated

2 shows about

100

5:

0.480

1511-l

to

those

cases,

the

of

control

the

23°C. The

thesize

observation

higher

that

levels

hypothesizing

controls is

36°C

800 x g supernatant

Table

by

strain

0.535

grown

at

%

1511

homogenate

percentage

of

of

g cells)

units/g

ml

cultures and 36°C

the

lost

enzyme

under

ization

of

both

the

mitochondria

(23)

beads.

Again,

pellet,

but

was

at

were

recovered

the

the

was the

level

in

with

sap.

An cell

not

GDH could

synthesized

out

involving

in

functions

syn-

be

in

the

and

that

explained

mitochondria, this

function

above.

carried

cell

used,

cytoplasmic

cytoplasmic

associated in

GDH activity was

and

mitochondrial

protein,

outlined

activity,not found

altered

nuclear

conditions

experiments

constantly

with

a regulatory synthesis

the

Further

was

of

that

cells

found

soluble

775

an attempt

to

nuclei,which, alternative

associated cytoplasmic

with fraction.

the

in

our

by

means

method

fragmentation

clarify

of

the

local-

experiments, preparation of

mitochondrial

of glass

Vol. 64, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table

2

Distribution of GDH activity in cultures grown at 23°C in the presence and absence and in cultures of the isogenic derivative

control

fraction

(2 g cells)

+erythr.

of 1511 strain of erythromycin, rho- 1511-1

(0.85

g cells)

rho-

(1.3

g cells)

units/g

%

units/g

%

units/g

%

homogenate

0.592

100

1.168

100

1.046

100

800 x g supernatant

0.536

90.5

1.040

89.2

0.968

83

800 x g sediment

0.056

9.5

0.126

10.8

0.184

17

This chondrial

result,

the conclusion with

together

functions

nuclei

reached is

with

do not

the finding

show enzyme

by other

localized

that

levels

investigators

in the

soluble

mutants

lower that

than

with

impaired

the enzyme not

cytoplasmic

mito-

the control,

phase

supports associated

and not

in the mito-

chondria. The biological suitable effective

mutants, tool

role with

of nuclear

GDH is

altered

nuclear

this

problem.

to tackle

still

unclear.

GDH activity,

The isolation

could

provide

an

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