Cell-free synthesis of succinate dehydrogenase and mitochondrial adenosine triphosphatase of sweet potato

Cell-free synthesis of succinate dehydrogenase and mitochondrial adenosine triphosphatase of sweet potato

Vol. 113, No. 1, 1983 May 31, 1983 AND BIOPHYSICAL BIOCHEMICAL RESEARCH COMMUNICATIONS Pages CELL-FREE 235-240 SYNTHESIS OF SUCCINATE DEHYDROGEN...

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Vol. 113, No. 1, 1983 May 31, 1983

AND BIOPHYSICAL

BIOCHEMICAL

RESEARCH COMMUNICATIONS Pages

CELL-FREE

235-240

SYNTHESIS OF SUCCINATE DEHYDROGENASE AND

MITOCHONDRIAL ADENOSINE TRIPHOSPHATASE

OF

SWEET POTATO Tsukaho

Laboratory

Hattori,

Yukimoto

Iwasaki,

and Tadashi

Asahi

of Biochemistry,

Faculty

Received

April

of Agriculture,

Nagoya 464,

Chikusa,

Shigeru

Sakajo

Nagoya University,

JAPAN

18, 1983

Polyadenylated mRNA was isolated from aged slices of sweet potato root The synthesis of tissue and translated in a wheat germ cell-free system. apoprotein of the flavoprotein subunit of succinate dehydrogenase and two of the subunits of mitochondrial adenosine triphosphatase were detected by indirect immunoprecipitation. The molecular weights of the immunologically identified products were 3,000 and 8,000-9,000 daltons larger than the mature flavoprotein subunit of succinate dehydrogenase and the mature subunits of adenosine triphosphatase, respectively. Most into

of the mitochondrial

mitochondria,

although

protein-synthetic

employed

synthesized

inside

studied

or outside

yeast

information

as well

into

(4-10). is

biogenesis.

When slices

of sweet

the mitochondria mitochondrial of which

the activity

organelles Yeast,

whether

(11). increases

Neurospora

Recently, of proteins higher

as to the

potato

root

during

site

tissue

accompanied Succinate

have

individual

mitochondria Concerning

proliferates enzymes

are synthesized

mitochondria.

available

as their

cell

(l-3).

to determine

from cytosol

using

little

the

apparatus

been mostly

the import

proteins

plant

their

and mammalian mitochondrial

of the

Abbreviations used: SDH, succinate dehydrogenase; subunit of succinate dehydrogenase; FlATPase, soluble adenosine triphosphatase; SDS, sodium dodecylsulfate;

and systems

proteins

detailed

mechanisms

have been

extensively

mitochondria,

are aged under

dehydrogenase

and imported

own genetic

of synthesis

by increases

aging

in cytoplasm

moist

conditions, of

one of the enzymes,

slices

(11,

SD flavoprotein formfP' of mitochondrial IgG, immunoglobulin 0006-291X/83

235

of

proteins

in the activities

tissue

are

however,

of their

(SDH) is

have

12).

The

G. $1.50

Copyright 0 19k3 by Academic Press, Inc. A/I rights of reproduction in any form reserved.

Vol. 113, No. 1, 1983 increase Sweet

can be suppressed potato

26,000

SDH is

and 65,000: subunit

aging

tissue

of the

chloramphenicol

composed

is

slices

and is

ribosomes

We used a different particularly enzyme, regulation

of the

mitochondrially

a wheat

sweet

root

potato

cytoplasmic

increase

in. activity

of

of the during

cycloheximide

or

was unexpected

because

DNA and synthesized

whether

sweet

on cytoplasmic

on

the effect

or import

of chloramphenicol the mitochondria

protein(s).

This

communication

germ cell-free

system

which

potato

ribosomes

into

tissue,

weights

indicates

the

like

the yeast

really

indicates

of the enzyme by a reports

and poly(A)+RNA synthesis

SDH,

from of this

the synthesis aged slices subunit

of

on

ribosomes. subunits

cytoplasmic

of FIATPase

ribosomes

the higher produced

The amount

by nuclear

to determine

synthesized

whether

produced

molecular

(13).

by either

(12).

15).

synthesis

of SDfp with

All

is

the

with

by chloramphenicol

approach

the SD fP' in other words,

with

to be coded

(14,

subunits

inhibited

Inhibition

RESEARCH COMMUNICATIONS

or chloramphenicol

a flavoprotein

increases

SDH has been reported

cytoplasmic

cycloheximide

of two different

(SDfp)

(12).

AND BIOPHYSICAL

by either

the latter

flavoprotein

yeast

BIOCHEMICAL

plant in

of mitochondrial

(l),

enzyme.

the cell-free proteins

have been

although

no evidence

We found system, is

established

that

the

which

actually

for

this

subunits

indicates synthesized

MATERIALS

to be synthesized

has been presented

of FIATPase that in

on

are

the cytoplasmic the cell-free

for

also origin

system.

AND METHODS

Roots of sweet potato (Ipomoea batatas Kokei No. 14) Plant Material. harvested in midsummer to autumn were stored at13-160C use. Slices of the parenchymatous tissue were incubated as described elsewhere (12). -Preparation of poly(A)+RNA. About 100 g of the tissue slices incubated for 1 day were hozgenized in a medium composed of 150 ml of 0.1 M Tris-HCl buffer (pH 9.0) coniaining 0.1 M NaCl, 2 mM Na2EDTA, 2 mM MgC12, and 1% (w/v) SDS, 150 ml of a mixture of phenol/chloroform/isoamyl alcohol [25/24/l, (v/v/v)] and 30 ml of 2-mercaptoethanol. The aqueous phase was washed twice with the phenol/chloroform/isoamyl alcohol mixture, then RNA was precipitated by adding one third the volume of 1 M NaCl and 2.5-fold the volume of ethanol and then allowing the mixture to stand at -20°C overnight. The precipitate was collected by centrifugation and washed twice with 70% (v/v) ethanol. The washed precipitate was dried and dissolved in 20 ml of 10 mM Tis-HCl buffer (pH 7.5) containing 0.5 M NaCl. The solution was applied to an oligo-dT cellulose (Collaborative Research Inc.) column. After the column had been washed with the same buffer, the bound RNA was eluted with 10 mM Tris-HCl buffer (pH 7.5). The poly(A)+RNA eluted from the column was precipitated with ethanol as described above, and the precipitate was dried and dissolved in a minimum volume of water. 236

BIOCHEMICAL

Vol. 113, No. 1, 1983

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Cell-Free Translation. Wheat germ extract was prepared by the method of Marcu and Dudock (16). The reaction mixture for the translation contained 20 mM Hepes-KOH (pH 7.5), 74 mM KCl, 1 mM MgC12, 0.6 mM spermidine, 1 mM ATP, 8 mM creatine phosphate, 5 rig/ml creatine kinase, 20 nM GTP, 1 mM dithiothreito 1, 50 nM each of 19 amino acids (excluding methionine), 2.5 mCi/ml [35S]methionine (1,100 Ci/mmol, New England Nuclear), and 150 rig/ml the poly(A)+RNA. The translation reaction was carried out at 3O'C for 1 h and stopped by adding 10% (w/v> SDS at a final concentration of 2%. Isolation of Sweet Potato %J- and QATPase and Preparation --of Their Antibodies. SDfp and its antibody were p--asdescribed before (12). The procedure for purification of FlATPase from sweet potato root mitochondria and preparation of its antibody will be described elsewhere (Iwasaki and Asahi, manuscript in preparation). IgG fractions were prepared from anti-SDfp, anti-FlATPase and nonimmunized sera as described previously (17). Indirect immunoprecipitation. Indirect immunoprecipitation of the translation products using protein A-Sepharose CL-4B (Pharmacia) was carried out according to the method of Watanabe and Price (18). SDS-Polyacrylamide Gel Electrophoresis and Fluorography. The antigenantibody complex bound toprotein A-Sepharoseeads was eluted with 30 nl of 0.125 M Tris-HCl buffer (pH 6.8) containing 4% SDS, 4% 2-mercaptoethanol and 40% glycerol. The beads were washed with 30 ~1 of water, which was then combined with the eluate. The combined solution underwent electrophoresis on a 10% polyacrylamide slab gel containing 0.1% SDS with the discontinuous buffer system as described by Laemmli (19). After electrophoresis, the gel was stained with Coomassie brilliant blue R, destained and then processed for fluorography using EN3HANCE (New England Nuclear). The dried gel was exposed to a medical X-ray film (Fuji RX, Fuji Phto Film, Tokyo) at -8OOC. RESULTS The activities including

SDH, increase

attain

maximal

Therefore,

values

1).

Several

but most

also

specifically

calculated confirm

that

Sweet

was really

IgG was performed

in

2). potato

and E) (Iwasaki

IgG.

3,000

the product

is

and Asahi,

slices

root

tissue,

10 h and tend slices

to

(12).

incubated

for

system

with

the poly(A)+RNA

immunoreactive

1 day,

Only one translation It

migrated

product

at a slower

rate

SD Its molecular weight fe' daltons more than the purified

immunologically

composed

of six

unpublished

data). 237

with different

as mRNA

to anti-SD

SD indirect immunoprecipitation fP' the presence and absence of an excess

The SDfp competed FIATPase

about of the

the tissue

were

IgG. fP the purified

than

for

incubation

cell-free

to anti-SD gel

potato

increasing.

products

to be approximately

antl-SDfP SDfp (Fig.

6'

in the

in sweet

lasting

from

to preimmune

reactive

phase

2 days during

translation

reacted

SDS-polyacrylamide

a lag

markedly

synthesized

was

enzymes

was isolated were

activities

SDfp

after after

poly(A)+RNA

when the

(Fig.

of mitochondrial

the translation subunits

Two translation

fP b$, was in

the

was SD fp' To with of purified product.

(a,

8, y, 6,

products

BIOCHEMICAL

Vol. 113, No. 1, 1983

Mrxlo-3

'

*

AND BIOPHYSICAL

3

RESEARCH COMMUNICATIONS

Mr~l0-~

'

*



.

g4-

i

94.

67-

:-..

67-

c

43-d"

43-C'

CL-

202

20-

0

144-

Fig. 1. Synthesis of the apoprotein of SDfp in a cell-free proteinsynthesizing system from wheat germ with poly(A)+RNA from sweet potato root of the immunoprecipitates from tissue as mRNA. Lanes 1 and 2, fluorograms 150 !.11 of the reaction mixture with control and anti-SDfP IgG, respectively; lane 3, electrophorogram of purified SDfp (stained for protein with Coomassie brilliant blue R). Electrophoresis and fluorography were done as described in MATERIALS AND METHODS. The gel was calibrated for molecular weights (Mr) with 14C-labelled marker proteins (Amersham): phosphorylase b (94,000). bovine serum albumin (67,000), ovalbumin (43,000), trypsin inhibitor (20,000) and lactoalbumin (14,400). Fig. 2. Competition of translation products with the purified SDfp for IgG was incubated with (lane antibody to SDfp. Twenty micrograms of anti-SDfp 1) and without (lane 2) 20 pg of the purified SDfp before being added to the of electrophoresis and fluorography are reaction mixture (75 111). Conditions given in Fig. 1.

reacted

specifically

products larger

to the

had apparent than

We suppose although

those that

the

sufficient

by 6,000

The present from wheat

molecular

to the purified

weights

of about

of the c1 and 6 subunits translation evidence

the cx and fi subunits larger

antibody

germ with

is

of yeast

and 2,000

work

products

shows

that

poly(A)+RNA

were

lacking.

FlATPase

daltons,

(55,000

FlATPase 63,000

Our proposition are

and 61,000

and 52,000,

precursors

synthesized

(Fig.

3). which

were

respectively).

of the cx and t3 subunits, is based as precursors

on the

fact

of sizes

respectively. DISCUSSION SD was synthesized in a cell-free system fP from sweet potato root tissue as mRNA. 238

These

BIOCHEMICAL

vol. 113, No. 1, 1983

AND BIOPHYSICAL

two-3

'

*

RESEARCH COMMUNICATIONS

3

94 67 -

AA

43-s

-Y

Fig. 3. Synthesis of FlATPase subunits in a cell-free protein-synthesizing Lanes 1 and system programmed with poly(A)+RNA from sweet potato root tissue. 2, fluorograms of the immunoprecipitates from 75 ~1 of the reaction mixture with control and anti-FLATPase IgG, respectively; lane 3, electrophorogram of the purified FlATPase from sweet potato, stained for protein. a, B, Y, 6, 6' Electrophoresis and fluorography were and E denote the subunits of FlATPase. carried out as described in Fig. 1.

Mitochondrial

mRNAs cannot

Therefore,

we inferred

ribosomes in

inhibitied

of

This

study

a larger

into

although

they

of slices

of sweet

or chloramphenicol

the mitochondria

(21,

22).

(12)

of SD is fP

potato

root

suggests

controlled

tissue

that

is

the

by a

protein(s).

like

to show that

(probably

precursors larger are

the

most mitochondrial

the subunits

daltons

germ system

SD is synthesized on cytoplasmic fP SDH (14, 15). Thus the fact that the increase

incubation

the first

in larger

8,000-9,000

yeast

in such a wheat

potato

cycloheximide

precursor

in yeast,

potato,

is

sweet

during

produced

synthesized are

fP

or import

mitochondrially

like

SD

with

by either

synthesis

in

that

as reported

the amount

be translated

2,000-6,000

of SD is synthesized fP Also, in sweet potato,

proteins.

a and fi subunits)

on the than

apoprotein

cytoplasmic

the mature daltons 239

of FIATPase

ribosomes.

polypeptides larger

are

The precursors in

in yeast.

the case of sweet There

has been

Vol. 113, No. 1, 1983 only

one report

of proteins second plant

for

in higher

to report

BIOCHEMICAL

AND BIOPHYSICAL

watermelon

malate

dehydrogenase

plant

mitochondria

(23).

--in vitro

translation

RESEARCH COMMUNICATIONS on the --in vitro

Thus,

of mitochondrial

this

synthesis

communication

proteins

using

is

the

higher

cells. REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Schatz, G. and Mason, T. L. (1974) Ann. Rev. Biochem. 43, 51-87. Schatz, G. (1979) FEBS Lett. 103, 203-211. Ades, Z. (1982) Mol. Cell. Biochem. 43, 113-127. Daum, G. and Schatz, G. (1982) J. Biol. Chem. 257, 13034-13041. Ohashi, A., Gibson, J., Gregor, J. and Schatz, G. (1982) J. Biol. Chem. g, 13042-13047. Suissa, M. and Schatz, G. (1982) J. Biol. Chem. 257, 13048-13055. Reid, G. A. and Schatz, G. (1982) J. Biol. Chem. 257, 13056-13061. Reid, G. A. and Schatz, G. (1982) J. Biol. Chem. 257, 13062-13067. Reid, G. A., Yonetani, T. and Schatz, G. (1982) J. Biol. Chem. 257, 1306813074. Daum, G., Gasser, S. M. and Schatz, G. (1982) J. Biol. Chem. 257, 13075-13080. T. Asahi (1978) in Biochemistry of Wounded Plant Tissues, ed. G. Kahl, Walter de Gruyter & Co., Berlin, pp. 391-419. Hattori, T. and Asahi, T. (1982) Plant & Cell Physiol. 2, 515-523. Hattori, T. and Asahi, T. (1982) Plant & Cell Physiol. 23, 525-532. Kim, I. C. and Beattie, D. S. (1973) Eur. J. Biochem. 2, 509-518. Lin, L. F., Kim, I. C. and Beattie, D. S. (1974) Arch. Biochem. Biophys. 160, 458-464. Marcu, K. and Dudock, B. (1974) Nucl. Acids Res. 1, 1385-1397. Maeshima, M. and Asahi T. (1981) J. Biochem. 90, 391-397. Watanabe, A. and Price, C. A. (1982) Proc. Natl. Acad. Sci. U.S.A. 2, 6304-6308. Laemmli, U. K. (1970) Nature 227, 680-685. Maccecchini, M. L., Rudin, Y., Blobel, G. and Schatz, G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 343-347. Leaver, C. J. and Gray, M. W. (1982) Ann. Rev. Plant Physiol. 2, 373-402. Fox, T. D. and Leaver, C. J. (1982) Cell, 6, 315-323. Gietl, C. and Hock, B. (1982) Plant Physiol. 70, 483-487.

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