Leucyl tRNA and leucyl tRNA synthetase in mitochondria of Tetrahymenapyriformis

Vol. 28, No. 5, 1967

Leucyl

8lOCHEMlCAL

tRNA and Leucyl

AND BIOPHYSICAL

tRNA Synthetase

RESEARCH COMMUNICATIONS

in Mitochondria

of Tetrahymena pyriformis

Y. Suyama and Joseph Eyer Joseph Leidy Laboratory Department of Biology University of Pennsylvania

Received July 25, 1967 Recently, Barnett, et al. (1967a, 1967b) reported that Neurospora mitochondria contain 18 different tRNA's and also mitochondria specific aminoacyl

tRNA synthetases.

tRNA, and their

corresponding

of Tetrahymena pyriformis. mitochondrial leucyl-tRNA mitochondrial.leucvl-tR.NA

Materials

We have isolated leucyl-tRNA By a partial

mitochondrial

synthetases,

from the ST strain

characterization,

and its synthetase and synthetase.

and whole-cell

both differ

we find

that

from the extra-

and Methods

An axenic culture of a strain of Tetrahymena pyriformis previously designated ST (Suyama, 1966) was used throughout these studies. Mitochondria were prepared as previously described (Suyama, 1966). The raffinose medium (0.2 M raffinose, 1 mM potassium phosphate pH 6.2, 0.25% bovine serum albumin) used in these studies and used either fresh or after These mitochondrial

was made with sterile storage at -20° C.

preparations

contain

little

double-distilled or no contaminating

material, as judged by plating out and by phase-contrast microscopy. mitochondrial DNA isolated from this mitochondrial fraction is not contaminated with nuclear DNA. RNA from the whole-cell homogenate was isolated

water,

Also,

by the phenol method

of von Ehrenstein and Lipmann (1961). Since this RNA contained rRNA and DNA, a RNA fraction eluted at about 0.5 N NaCl from a methylated albumin coated Kieselguhr (MAK) column (Sueoka and Yamane, 1962) was collected. This tRNA fraction was then dialysed against double-distilled water and lyophilized to a powder, which was stored at -200 C. until used. This RNA is estimated to contain less than 1% of mitochondrial tRNA. The tRNA from the mitochondrial fraction was prepared as follows. The washed mitochondria were suspended and lysed in 0.01 M acetate buffer pH 5.0

‘746

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 28, No. 5, 1967

and 2% sodium phenol

dodecyl

treatment

fractioned

as the

preparation

following

whole-cell

of the

methods

were

6 min at 500 xg, to remove

and the

prepared

dialysed

pH 7.4,

0.01

alumina at

'mitochondrial'

enzyme after

10,000

The two tRNA's Nuclear

Corp.,

in the

following

10 bmoles

sp.

amounts

of

aliquot

was taken,

tRNA.

and the

radioactivity

reached

within

incubation

at

sodium

phosphate

phenol

were

then

centrifuged.

remove

phenol,

counting

of

KCl,

H3-L-leucine, After

various

to 0.13 times

and precipitable determined. incubation.

37O C.,

5 ml of

buffer

added.

and then

fractionated

homogenized

by

The homogenate used

as the

(New England

Tris-HCl, pH 7.5, pmole C14-L-leucine extract

and varying

at 37O C., the

ml

maximum activity

charged

tRNA,

buffer

volume in the

0.1

on a membrane

filter was

after

(0.3

N NaCl,

of water-saturated cold

for

was extracted Tricarb

20 min and with

by MAK chromatography. in a Packard

a

5 ml

(NEN, 170 mc/mmole)

saline-phosphate

supernatant

Cl4 and H3 was done

(about

supernatant

collected

isolating

was shaken

The resulting

C.

was then

incubation

and an equal

mixture

was

C.

In any case,

pH 6.7)

This

M TrisiHCl

by resuspending

ml of enzyme

of

For cold

-70°

C14-L-leucine

material

thus

(0.01

buffer

50 pmoles ATP, 0.006

1 pmole

0.10

5 min

at

6,000

in a Spinco

buffer

or H3-L-leucine

mixture:

6 min at

homogenizer.

either

155 mc/nnnole)

10 pmoles

which

-70°

for

supernatant

was prepared

at

with

1 ml reaction

MgC12, pmole

charged

act.

and stored

10 min and the

storage

the

The dialysate

in a Teflon-glass

xg for

were

and the

Tris-magnesium

protein),

powder

was centrifuged

xg,

for

centrifuged

in Tris-magnesium

100 mg mitochondrial

with

tRNA synthetases,

centrifuged

fraction

pellet

obtained

was centrifuged

was then

105,000

synthetase

mitochondrial

per

further

enzyme

same

RNA thus

2-mercaptoethanol).

mitochondrial"

The mitochondrial twice-washed

acyl

homogenate

against

1 ml/liter

the

a tRNA fraction.

supernatant

extensively

"post

grinding

supernatant

to

and the

amino

whole-cell

120 min at

M MgC12,

designated

or 0.024

homogenate, yielding

This for

was subjected

corresponding

used:

mitochondria.

L centrifuge

buffer

The lysate

by MAK chromatography,

For

model

sulfate.

liquid

ether

Differential scintillation

counter.

Results

and Discussion When mitochondrial

and post-mitochondrial patterns while

such as Fig. whole-cell

and whole-cell enzyme

lA and Fig.

tRNA shows

tRNA's

fractions

1B result.

two equally-charged

747

are charged

respectively, We find regions

by mitochondrial

MAK chromatography repeatedly for

that

C14-leucine,

to

xg

Vol. 28, No. 5, 1967

BIOCHEMICAL

FRACTION

Figure

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

NUMBER

1 MAK column chromatography of mitochondrial (A) and-whole-cell (B) charged with mitochondrial and post-mitochondrial enzymes, leucyl-tRNA's, respectively. A: 2.5 ml of the standard reaction mixture (see methods) containing 200 ng of mitochondrial tRNA and 0.50 ml of mitochondrial extract were incubated for 20 min at 37O. RNA was re-purified as described, applied to a MAK column, and eluted with a linear salt gradient made with 130 ml each of 0.3 N NaCl and 0.9 N NaCl, sodium phosphate buffer (0.05 M, pH 6.5). The continuous OD260 profile was obtained by monitoring the effluent through a flow cell (path length l/volume = 10 mm/O.50 ml) C14-leucyl in a Beckman DB spectrophotometer with an attached recorder. tRNA was precipitated by trichloracetic acid (10%) in the cold from 3 or 4 ml fractions and collected on membrane filters by suction. The filter papers were dried and radioactivity was counted in a liquid scintillation spectrometer. B: 1.0 ml of the reactive mixture containing 380 pg of All operations whole-cell tkNA and 0.26 ml of post-mitochondrial enzyme. were the same as (A), except an additional 400 ng of the tRNA was added as CD260 marker before MAK column fractionation.

mitochondrial these

tRNA is

Two explanations from

might

both

Cl4 -1eucine

while extracts amounts

the

each

account

mitochondrial

post-mitochondrial

synthetases,

lacks

with

largely

in only

one of

regions.

isolated the

charged

enzyme capable

mitochondrial

for

these

extract

of enzyme

To clarify

these

contains

region only

If

the

tRNA's

same,

leucyl-tRNA

of amino-acyl

one.

then

(2)

If

tRNA,

both

whole-cell tRNA contains equal while mitochondrial tRNA

we performed

748

(1) are

two independent

a different

extract

points,

lysates

contains

charging

contain identical synthetases, then of at least two kinds of leucyl-tRNA, one.

observations.

and whole-cell

the

following

cross-

Vol. 28, No. 5, 1967

charging

experiments.

enzyme;

and,

enzyme.

that being from

of

resulting

in the

mitochondrial

able

whole-cell

one peak

with

mitochondrial MAR

Neither

whole-cell

tRNA peaks;

tRNA peak.

differs

mainly;

post-mitochondrial

independent

tRNA's.

C14-leucyl

tRNA by lacking

from

C14-leucine

synthetase

to charge

results

C14-leucyl equal

second

with

tRNA was charged

2B show the

tRNA showed

defective (1)

the

RESEARCH COMMUNICATIONS

tRNA was charged

whole-cell

2A and Fig.

mitochondrial

largely

Mitochondrial

independently,

Fig.

chromatography nor

BIOCHEMICAL AND BIOPHYSICAL

from

both

These

results

post-mitochondrial

of

are

suggest synthetase,

and (2) mitochondrial

one region

tRNA

profiles

tRNA differs

leucyl-tRNA. CPM

CPM A

400

I&

CPM

FRACTION

Figure

2 MAR column chromatography of leucyl tRNA's, cross-charged with enzymes respectively. A: 2.5 ml contained 200 hg of mitochondrial enzyme. All other operations and B: 1 ml reaction mixture containing of mitochondrial enzyme was used. were the same as in Figure 1B.

At

this

point

peak

of mitochondrial

cell.

Mitochondrial

and whole-cell were (Fig.

NUMBER

we attempted

tRNA,

charged

co-chromatographed 3) indicate

suggesting

to distinguish

tRNA from tRNA, after

a significant

a corresponding

mitochondrial (A) and whole-cell (B) post-mitochondrial and mitochondrial of the reaction (see methods) mixture tRNA and 0.50 ml of post-mitochondrial conditions were the same as in Figure 1A. 380 pg of whole-cell tRNA and 0.26 ml All other conditions and operations

the

charged with

with

H3- leucine

the

by post-mitochondrial between

in mitochondrial

749

of

by mitochondrial

The results

difference

leucyl-tRNA

leucyl-tRNA

C 14-leucine

isolation. difference

further

corresponding

of the

this

peaks

the whole enzyme, enzyme,

experiment of H3 and C14,

and whole-cell

tRNA's.

Vol. 28, No. 5, 1967

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

SO0

400

200

40 FRACTION

30

35 NUMBER

25

cl4 -leucyl-tKNA and whole-cell Figure 3 Co-chromatography of mitochondrial H3-leucyl-tKNA charged with mitochondrial and post-mitochondrial enzymes 2.5 ml of the reaction respectively. Two reaction tubes, each containing but one with 200 pg mitochondrial tKNA, C14-leucine, and 0.50 ml of mitochondrial enzyme and the other with 380 p,g whole-cell tKNA, H3-leucine and 0.50 ml of post-mitochondrial enzyme, were separately of tKNA's, both incubated at 370 for 15 min. After re-purification Input tKNA's were mixed and co-chromatographed through a MAK column. count ratio of H3 : Cl4 was 2:l.

In these analyses,

the radioactivity

profile

of mitochondrial

tKNA shows a minor peak coinciding with the first-eluted H3-leucyl tKNA. This indicates that our mitochondrial contain

this

material

C14-leucyl

peak of whole-cell tKNA preparations

in much reduced amount.

Although the available evidence suggests that mechanisms of protein synthesis in mitochondria are analogous to those of bacterial systems, it is premature at present to make any conclusion as to the funtional role of the mitochondrial tKNA's, particularly in light of the soluble amino acid incorporating systems existing in E. +J (Kaji, e_t al. 1965) as well as in mitochondria (Kalf, 1964). Furthermore, the origin of mitochondrial tKNA is an open question. The results of DNA-RNAhybridization studies (Suyama, 1967) made with the tK.NA fraction of Tetrahvmena are more compatible with the idea that these tKNA's are transported than made within the mitochondrion.

750

into mitochondria

BIOCHEMICAL

Vol. 28, No. 5, 1967

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Acknowledgement This Atomic

investigation

Energy

Literature

cited

Barnett. (1967a).

W. E.,

Barnett, 57:1775

W. E., (1967b).

Kaji, Kalf,

A.,

and D. H. Brown. D. H. Brown,

H. Kaji,

G. F.

was supported

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Biochemistry. and T. Yamane.

Suyama,

Y.

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Y.

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and G. D. Novelli.

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and J.

Sueoka, (1962).

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by a grant

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Commission.

U. S. 57:452

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751

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the