Vol.

64,

No. 2, 1975

BIOCHEMICAL

A TEMPERATURE

AND

SENSITIVE

BIOPHYSICAL

MlTOCHONDRlAL

SACCHAROMYCES E.M.

Received

March

SUMMARY: mutant of yeast an interaction biochemical

31,

those

50 x lo6 genes

(l),

coding

mitochondrial previously MATERIALS

J. Marmurd

of Genetics* and Biochemistry+ College of Medicine, Bronx,

synthesized chain

are

the

could

code

for

novel

mutations

N.Y.

(l),

species

mitochondrial a number

are

genome

be inferred

in tempemture

of this-class

(2)

While known

with

of additional

could

resulting

METHODS

however,

tRNA

functions

AND

function;

is uncertain.

for

mutation

sensitive

polypeptides

and

mitochondrial

rutamycin

these

yeast

reported

of the

for

rRNA

DNA,

components

essential

specifying

mitochondrial

mitochondrial

and

inherited temperature sensitive respiratory Detection of nuclear suppressor mutations nuclear and mitochondrial genomes. A preliminary is presented.

between the characterization

of genes for

OF

A mitochondrially has been isolated.

respiratory

location

MUTATION

1975

Mitochondrially of the

COMMUNICATIONS

CEREVISIAE

Storm*

Departments Albert Einstein

RESEARCH

is further

the genes,

weight

Mitochondrially

described

e.g.,

of

carrying

deficiency. in this

with

located

of strains

respimtory

and

cellular

to be associated

by the analysis sensitive

ATPase

a few

a molecular

products.

deficient indicates

A

paper.

Strain 2-38 (5-6) C (a/a leul/Teul ural/uml + /ade2 trp5/ + /ma1 1 f /sue 1 +/serADE 15/+ TOLlsI), obtained f&n E.Jones,was mutagzized wx N-mexylNTn itro-N-nitrosoguanidine (5mg/ml) for 5 min. at 23’C. The cells were washed free of the mutagen and plated at 230 on enriched medium (1% yeast extract plus 2% peptone) containing 3% glycerol and 1% ethanol (YPGE) and oligomycin (5pg/ml). Single colony isolates of antibiotic resistant clones were replica plated onto YPGE medium and incubated at permissive (23O) and non-permissive (360) tempemtures. Clones temperature sensitive on YPGE were mated with HK15 (*l/lysl) obtained from G. Fink, and the resulting tetraploids were screened for somatrc segregation of the temperature sensitivity.

YPGE

= medium and 2%

containing agar.

1%

yeast

extract,

2%

peptone,

3%

glycerol,

1% ethanol

Vol. 64, No. 2, 1975

Sparuiation

BIOCHEMICAL

of tetraploid

clones. temperature

ceils

A temperature sensitive

which

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

exhibited

somatic

segregation

Spontaneous revertants of the temperature sensitive obtained by selecting clones of haploid derivatives incubated on YPGE plates at 36’ for 5 days.

were were

gave

rise

sensitive % diploid clone was finally sporulated haploid clones of which aa53-2d-a is representative.

to diploid

to yield

respiratory deficient of aa which grew

mutant after cells

Cytoplasmic inheritance was determined by standard procedures of somatic segregation and tetrad analysis (3) in crosses with D6 (I( arg met), a respiratwy sufficient strain obtained from D. Wilkie. Respiratory defizenrderivatives were after growth in ethidium bromide (lOug/ml) at 23O for 18 hrs (4). The resulting respiratory deficient stmins, aa 2d-a EB and D6EB, lack detectable mitochondrial as determined by analytical cesium chloride density gmdient centrifugation.

obtained DNA

Enzyme activities were assayed in lysates or crude mitochondrial fractions prepared from cells grown to stationary phase in broth containing 1% yeast extmct, 2% peptone, and 1% galactose. Cells were washed and resuspended in buffer containing 0.25M sucrose, 10 mM N-2 hydroxyethylpiperazine-NT -2 ethanesulfonic acid, 1 mM EDTA, pH 7.3 then shaken for 35 sec. in a Braun homogenizer in the presence of glass beads (0.45-0.50 mm). Activities were determined in the supernatant fraction present after 10 min. centrifugation at 1085 x g. Alternatively a crude mitochondrial pellet was obtained after an additional 10 min.centrifugation at 1085 x g followed by a 20 min.centrifugation at 12,100 x g. Standard spectrophotometric methods were used to assay NADH: cytochrome c reductase (EC 1.6.2.1) (5), succinate: cytcchrome c reductase (5), cytochrome c oxidase (EC 1.9.3.1) (6) rutamycin sensitive ATPase (EC 3.6.1.3) (7) activities and for protein determination (8). RESULTS Growth enriched

medium,

in Fig. used

curves

1.

Growth

as a carbon

confers

at room The

the

is observed

Sporulation

at either

on glucose

or ethanol, at 23’

temperature.

medium,

but

strain

at 23’

the

deficient

the

deficient and

not at 36’;

Thus

respiratory

of inheritance

of these

mutant

diploids was

of the

of mutant

sensitive

phenotype

glucose

respiratory

strain

strain

at 36O are

however, carries

phenotype. retained

haploid

its ability

presented

glucose a mutation

In addition,

in

can which

after

to grow

be

72 hours

on YPGE

tempemture.

population

temperature

mutant

in ethanol

sensitive

pattern

heterogeneous

sensitive

either

source

at 36’

temperature

containing

a temperature

of growth plates

of the

observed

mutation

and with

resulted (Table

wild

was type

diploids

a respiratory in tetrads 1).

determined

was obtained

competent in which

In addition,

as follows:

0:4

tester and

sporulation

4:0

A from

strain

crosses

(Table

segregation of diploids

of

1). of the having

the

Vol.

64, No. 2, 1975

BIOCHEMICAL

AND

0

24

I

BIOPHYSICAL

I

I

RESEARCH

1

I

1

COMMUNICATIONS

1

A8

TIME biourr)

Figure

1.

Growth at two

of the different

mutant strain temperatures.

Overnight cultures grown at room broth containing either 2% glucose

aa53-2d-a

as a function

temperature in or 2% ethanol

of carbon

source

1% yeast extract 2% peptone as a carbon source were used

to innoculate cultures grown in these media at either room temperature or 360. At various times, aliquots of cells were removed, diluted and plated on enriched medium containing 2% glucose. Plates were incubated at room temperature and colonies were counted three days after plating. 0, growth at room temperature, 2% glucose; 8, growth at 36o,2% glucose; A, growth at room temperature, 2% ethanol; A, growth at 36O, 2% ethanol.

mutant derived and

type

phenotype from meiotic

resulted

diplaids

thep’

diploids

and

was crossed

with

and

tetrads

mutant

be associated The

having

segregation

When

with mutant

P O=respiratory

in 0:4

derivative

D6EB, were

are

type

deficient

sufficient

consistent

tetrads

with

observed

wild

type

phenotype.

Both

of the

cytoplasmic

inheritance.

was crossed

were

a respiratory

was

while

aa53-2d-aEB

mitochondrial stmin

segregation

a respiratory

patterns

wild

meiotic

observed

deficient (Table

(Table p

1).

Thus

’ tester the

only

with

the

1).

In contmst,when

stmin,

tester

tetrads

only

cytoplasmic

above

strain,

mitotic

only

wild

aa53-2d-a

mutant

mutation

were

diploids appears

to

DNA. found

yeast

to revert

stmin

with

lacking

754

a frequency

detectable

of

10

-8

mitochondrial

. Two

classes

DNA

of

Vol. 64, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

HS A.

HS L

I+

IO

I+10

min +I

I+

min -+I

min -+I

ItlO

t-nit-8 -+I

IO

E

I+-

IO min

-+I

l-10

min

G“‘I” ILi L:

HS

ZOJJM

Cyt.E

c

A

k-10

min

-I

-4

I+lOmin--rl

762

Vol.

64, No. 2, 1975

spontaneous

BIOCHEMICAL

revertants

have

Mendelian

inheritance.

mutation

is an interesting

products.

This

Members

reversion

at the Further

of the original

synthesis

mutant

and

mitochondrial

strains

(Table

2).

mutant

at both

at 36’.

Further

details

In studies

of the

of nuclear

exhibit

class

COMMUNICATIONS

exhibit

temperature

dominant

sensitive

and

mitochondrial

may

mitochondrial

mitochondrial

gene

inheritance,

be summarized

as follows:

synthesis

present

from

mutant

from

of studies

at 36’

fractions

and

isolated

of the

grown

these

to five-fold

fractions

non-permissive

cells

c reductase three

were

and

In addition,

In contrast, obtained

interaction

permissive

activity.

were

of one

of the

DNA

derived

activities

Members

RESEARCH

probably

due

to

site.

in succinate:cytochrome

levels

BIOPHYSICAL

suppression

class

mitochondrial

c oxidase

reduction

second

of the

fractions

cytochrome

of the

mutant

and

parent

isolated.

nuclear

case

analysis

protein

been

AND

Mitochondrial at comparable

levels

temperatures.

However,

were

found

exhibited

c reductase

reductions

in rutamycin

from

mutant

mutation

and

to lack

a significant

NADH:cytochrome

either

in

sensitive

or parent

revertants

will

ATPase

strain

appear

grown

elsewhere

W

DISCUSSION

in this

paper

common

has the

class

significant

advantage

appear

phenotype. recently

that

Another affect

the

limited

discovered

mitochondrial

mutations,

of cytochromes

More

alteration

the

b,

petite

and

c,

mutation

phenotype.

accompanied

loci

respiratory

In contrast,

the

exhibits

by a lack

of mitochondrial

could DNA

described

mutation,

respiratory

of mitochondrial inherited

loss or

competence produce might

protein

(1,9).

the

Thus,

petite

be expected

deficient

stmins

results

in resistance

in the

which

retain

(10).

of mitochondrially

mitochondrial

the

or loss of many

alterations

synthesis

i.e.,

3,

genome,

well-defined

DNA a +a

mitochondrially

protein class

mitochondrial

of mitochondrial

absence

it would

of the

of a stable,

of mitochondrial

alteration

synthesis,

which

function

function.

inherited The

mutation

use of such

756

strains

is a potentially

to antibiotics promising

room

temperature,

(+)

2-38(5-6)C

RT:

(+)

(tj

act53

at 24OC.

2-38(5-6)C

(ts) -

a053

Strain

assayed

mitochondrial

crude

(ts)

c

and

parent

cytochrome at two

23-250C.

36

0.394

0.560

0.?80

RT

0.412

0.152

(pM cykhrome mg-’ protein)

cytochrame reductase

Succinate:

grown

0.0024

(+)

NADH:

0.000

strains

c reductase,

36

(PM _clytoch;ome c oxidized min mgprotein)

Cytochrome oxidase

mutant

succinate:

0.354

from

c oxidose,

RT

(OC)

temperature

obtained

of cytcchrome

fractions

Growth

Activities

2.

Table

c

Fractions

c reductase

7.812

0.810

0.368

0.610

min

-1

NADH: c ytoc hrome reductase c reduced

temperatures.

cytochrome

c

were

in

Vol.

64, No.

probe

BIOCHEMICAL

2, 1975

for

the

study

of the

relationship

between

demonstrated

(9).

While mutation

resistance

a preliminary

has been

should

gene-protein mutation

should

the

a mutant.

be useful

in the

provide

intemction

some

presumably

of the

interaction

than

one

a point

into

the coded

to tempemture

gene

sensitive,

and

functional

and

Jeffrey

Lam

for

map

deficient

mitochcndrial

enzyme,

in a single

mutants

problem

detailed

of a mitochondrial

mutation

of mitochondrial

insights

a genetic

more

represents

mitochondrially

leading

to construct

it affects

and

of organelle

their

Mutations

respiratory

deficiency

mitochondriolly

the

gene.

suppressors

biogenesis

products.

of the

to be

sensitive

respiratory

definition

of the

remains

a more

sensitive,

of a clearcut

nature

temperature

presents

temperature

the

product

inherited paper

COMMUNICATIONS

however,

translation

this

formulation

and

and

genes

This

Although

interesting

of nuclear

mitochondrial possible

study

a mitochondriai

(1 l),

RESEARCH

genes,

of a mitochondrially

of such

is revertable,

In addition,

and

by others

relationship.

BIOPHYSICAL

of mitochondrial

report

published

characterization mutation

function

AND

and

the

in other will

make

located

genes

it in

yeast. We

thank

assistonce,

and

This Elizabeth

William Dr.

research M.

Genetics,

GM

Storm

Perry

Keng-Bon

Chinsky

helpful

was supported

by grants

was supported

by on

their

excellent

technical

discussion. CA

NIH

for

12410

Training

and Grant

GM

19100 to the

from Department

NIH. of

00110.

REFERENCES

:: 3. 4. 5. 6.

Borst, Storm, Coen,

P. (1972). Ann. Rev. Biochem. 41:333-376. E.M., Lam, K-B., and Marmurz. (1974). Genetics 77:s62. D., Deutsch, J., Netter, P., Petrochilo, E.,ond Sloniai, P. (1970). Sot. Exp. Biol. Symp. 24:449-496. Goldring, E.S., GrossmanTL.I., Krupnick, D., Cryer, D.R., and Marmur, J. (1970). J. Mol. Biol. 52:323-335. Schotz, G. and KTma, J. (1964). Biochim. Biophys. Acto 81:448-461. Whorton, D.C. and Tzagoloff, A. (1967). In Methods in Enzymology, Vol. X, R.W. Estabrook and M.E. Pullman, eds.lew York: Academic Press, p. 245-250.

758

Vol.

7. 8. 9. 10. 11.

64, No.

2, 1975

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Tzagoloff, A. (1969). J. Biol. Chem. 244:5020-5026. Lowry, O.H., Rosebrough, N.J., Fart--.L., and Randall, R.J. (1951). J. Biol. Chem. 193:265-275. SchX, G. and Mason, T.L. (1974). Ann. Rev. Biochtm. 43:51-87. Flury, U., Mahler, H.R., and Feldman, F. (1974). J. Biomhem. 249:6130-6137. Handwerker , A., Schweyen , R.J., Wolf, K.,and Kaudewitz, F. (1973). J. Bacterial. 113:1307-1310.

759